Systems, compositions, and methods for transplantation and treating conditions

ABSTRACT

Systems and methods for purification and concentration of autologous alpha-2 macroglobulin (A2M) from whole blood and or recombinant A2M are provided. Also provided are methods of treating wounds with A2M. Methods for utilizing A2M in combination with other treatments (e.g., platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half-life of the protein for treating wounds.

CROSS-REFERENCE

This application is a divisional application of U.S. Nonprovisionalapplication Ser. No. 14/471,663, filed Aug. 28, 2014, which claims thebenefit of U.S. Provisional Application No. 61/871,009, filed on Aug.28, 2013, U.S. Provisional Application No. 61/990,522, filed on May 8,2014, and U.S. Provisional Application No. 61/990,524, filed on May 8,2014, all of which are incorporated herein by reference in theirentirety.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 25, 2016, isnamed 37151_703_401_SL.txt and is 85,687 bytes in size. Theaforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND

The physiological cellular response to tissue injury in the skinprogresses through a sequence of structured phases and normally resultsin a nearly complete recovery the injured area. Wounds can be eitheracute or chronic with respect to healing. In chronic wounds, theduration of the wound healing processes is either much slower or static.Wound healing depends on several factors, including the patient's ageand physical condition, the location of the wound, the cause of theinjury, and accompanying diseases such as diabetes or renalinsufficiency, which all have a negative effect on wound healingprocesses.

Wound healing involve several populations of cells (thrombocytes orplatelets, neutrophile granulocytes, macrophages, fibroblasts, andkeratinocytes), soluble factors (cytokines and growth factors), andproteases (e.g., matrix metalloproteinases [MMPs], plasmin, andelastase). Healing initially involves hemostasis initiated by theactivation of the clotting cascade. Fibrin clots forming the provisionalwound matrix entrap erythrocytes and platelets and block blood flow.Numerous growth factors (e.g., platelet-derived growth factor (PDGF),platelet-derived angiogenic factor (PDAF), transforming growth factorand epidermal growth factor (EGF)) are released from platelet granulesand chemotactically attract neutrophils, fibroblasts, endothelial cells,and keratinocytes into the wound. The initial release of growth factorsfrom platelets is important in initiating the phases of wound healing.

Inflammation is the initial response to tissue injury. Within 6 h aftertissue injury, inflammation begins. The main goal of the inflammatoryphase is to provide rapid hemostasis and begin the sequence of eventsthat leads to regeneration of tissue. Neutrophil granulocytes typicallyappear in wounds first and control bacterial contamination and cleansethe wound from cell detritus. After 48 h, the concentration ofneutrophil granulocytes is maximized. Monocytes begin infiltrating thewound site 24 h after injury, attracted by chemotactic factors includingcomplement factor 5, fibrin degradation products, and TGF-β. In responseto wound cytokines, monocytes differentiate into wound macrophages toaid wound repair.

During the proliferative phase, the damaged, necrotic tissue that isbeing removed via phagocytosis starts to be replaced with living tissuethat is specific to the local tissue environment. Proliferation isprimarily characterized by granulation tissue. MMPs take part in thestructured development of granulation tissue by removing damaged matrixproteins, helping cells migrate into the wound, and developing new bloodvessels.

About 2 days after injury, macrophages from monocytes begin expressinggrowth factors. Macrophages continue to release PDGF, macrophageangiogenesis factor, and TGF-β. PDGF, macrophage angiogenesis factor,and angiotensin stimulate new blood vessel formation, generatinggranulation tissue in the wound. EGF, keratinocyte growth factor, andPDGF stimulate epidermal cells to migrate, divide, and differentiate(keratinize), covering the granulation tissue with a cellular barrier todesiccation and infection.

During remodeling, newly generated tissue reshapes and reorganizes tomore closely resemble the original tissue. Remodeling begins about the7^(th) day of wound healing and can continue for 6 months to a year.Early in the remodeling, the provisional wound matrix, predominatelyfibrin and fibronectin, is replaced with proteoglycan molecules andcollagen molecules (type III, type I) that become cross-linked byenzymatic action, which greatly increases the tensile strength of thescar matrix. In addition, some fibroblasts are stimulated to transforminto myofibroblasts that contract the wound matrix. The high density ofnew blood vessels and myofibroblasts in the scar then decrease asvascular endothelial cells and fibroblasts undergo apoptosis, and thehypertrophic epidermal layer becomes thinner. At the end of the woundhealing process, the wound is closed. However, the repaired tissue doesnot completely regenerate the original tissue structure, and some levelof functionality of the scar tissue is usually lost.

Platelets play a prominent role as one of the first responders duringthe acute inflammatory phase. In response to tissue damage, plateletsare activated resulting in the formation of a platelet plug and bloodclot for hemostasis. The alpha granules of activated platelets containnumerous proteins that influence wound healing. Thrombin productionultimately occurs and converts fibrinogen to fibrin which binds toplatelet surface receptors. Proteins from platelet degranulation arepartly responsible for cellular chemotaxis, proliferation, anddifferentiation. This includes removal of tissue debris, angiogenesis,establishing the extracellular matrix, and regeneration of theappropriate type of tissue.

Platelet rich plasma (PRP) contains clotting factors and higherconcentration of platelets than baseline. The portion of plasma thatremains deficient in platelets is known as platelet poor plasma (PPP).PPP has clinical roles as fibrin sealant for hemostasis.

A common characteristic of chronic wounds is elevated proteaseactivities. Thus, local (or systemic) treatment of chronic wounds withprotease inhibitor(s) could promote healing. However, high levels ofprotease activity in chronic wounds of widely differing aetiology havebeen shown and may be related to a problem with the healing processitself rather than with the aetiology of the wound.

MMPs play vital roles in initial wound debridement as well as in thephases of angiogenesis, epithelialization, and scar remodeling. Abalance between proteases and their inhibitors is necessary for acorrect wound healing, and elevated levels of proteases and reducedlevels of inhibitors have been found in chronic wounds. Increased levelsof MMP-2 and MMP-9 have been demonstrated in various chronic woundliquids. Increased levels of MMP-1 and MMP-8 have been found indecubital ulcers, and MMP-13 in venous ulcer lesions. Reduced levels ofTIMPs have been found in chronic wound fluid. The MMP-9 to TIMP-1 ratiomay be a predictor for chronic wound healing, as an inverse correlationwith the healing tendency of chronic pressure ulcers has been shown.(Ladewig et al).

Similar processes may occur in non-healing or poorly healing diabeticfoot lesions. Loots et al., Dahn et al., Mansbridge et al.). Higherconcentrations of MMPs and reduced concentrations of MMP inhibitors havebeen found in diabetic wounds compared with trauma lesions of a controlgroup. Unlike normal wound healing, an overexpression of these proteasesseems to support a delayed wound healing and lead to a failure of woundsto heal. Additionally, an imbalance between MMPs and TIMPs thatcontributes to the pathogenesis of nonhealing chronic lesions may exist.Chronic diabetic foot ulcers have been treated with the antibioticdoxycycline, which is also a competitive inhibitor of certainmetalloproteases. Dressings that contain high concentrations of gelatin,which is a substrate for MMPs, have also been used. Elastase and plasminactivities in wound fluids have been found at significantly reduced by alocal therapy with Promogran, which may improve healing by reducing theactivities of MMPs in the molecular environment of the wound. Cullen etal. A dressing consisting of metal ions and citric acid has also beenused and reduced reactive oxygen species and MMP-2 production in vitro.

Use of recombinant PDGF (Regranex) for diabetic foot syndrome showedimprovements in the probability of healing and reduction of healingtime. Smiell et al. It was also determined that the wound bed needed tobe properly debrided for the growth factor to have maximum benefit.Thus, wound bed preparation is important, and emphasizes the removal ofbarriers to healing and the integration of advanced technologies inwound care.

Alpha-2-macroglobulin (A2M) is a highly conserved protease inhibitorpresent in plasma at relatively high concentrations (0.1-6 mg/ml). It isunique in its ability to inhibit all the major classes of proteases(Bhattacharjee et al., J. Biol. Chem. 275, 26806-11 (2000)). A2M can beproduced by several cell types, such as hepatocytes, lung fibroblasts,macrophages, astrocytes and tumor cells (Borth W, Ann N.Y. Acad. Sci.737:267-72 (1994)). A2M often exists as a tetramer of four identical 180kDa subunits that forms a hollow cylinder-like structure. It can presentmultiple target peptide bonds to attacking proteases in its central“bait” domain. A2M can be the major protease inhibitor acting on foreignproteases, such as snake venoms. However, there are many other proteaseinhibitors in the circulation and it has been proposed that A2M can haveother functions including binding to and regulation of cytokine andgrowth factor activity, promotion of tumoricidal capabilities ofmacrophages, and enhancement of antigen presentation. A2M can also be atargeting carrier for cytokines or growth factors.

Despite advances in the understanding of the principles underlying thewound healing process, there remains a significant unmet need forsuitable therapeutic options for wound care and tissue repair andimproving and/or promoting wound healing, including wounds that do notheal at expected rates, such as delayed-healing wounds, incompletelyhealing wounds, and compromised wound healing such as is seen in chronicwounds, scarring and abnormal or excessive scarring, including keloidand hypertrophic scarring, atropic scarring, widespread scarring, andscar contractures, as well as adhesions including surgical adhesions.There is a need in the art for improved methods and compositions fortreating conditions such as those caused by acute and chronic wounds,inflammation, fibrosis, scarring, and adhesions.

Therefore, it is an object of the invention to provide compositions,systems, methods, and kits for the detection, diagnosis, and treatmentof inflammation, degradation of extracellular matrix, and wounds. It isanother object of the invention to provide systems and methods toproduce compositions for the treatment of inflammation, degradation ofextracellular matrix, and chronic wounds. It is another object of theinvention to provide biomarkers and methods for identifying sites ofchronic wounds. It is another object of the invention to provide methodsfor diagnosing or assisting in the diagnosis of the presence ofpathologies that are causative of chronic wounds. Yet another object ofthe invention is to provide biomarkers and methods to determine anappropriate therapy for a subject experiencing chronic wounds. Anotherobject of the invention is to provide biomarkers and methods to monitorand assess the efficacy of a treatment for chronic wounds. Anotherobject of the invention is to provide compositions and methods fortreating chronic wounds and for selecting treatment sites and methodsfor treatment of chronic wounds.

It is another object of the invention to provide variant polypeptidesfor treating chronic wounds. It is another object of the invention toprovide variant A2M polypeptides with a higher protease inhibitoryactivity than a wild-type A2M polypeptide. It is another object of theinvention to provide methods of making variant polypeptides for thetreatment of chronic wounds.

SUMMARY OF THE INVENTION

One aspect provided is a method for the treatment or prophylaxis of awound in a subject, comprising administering to the wound an effectiveamount of a composition comprising alpha-2-macroglobulin (A2M) isolatedfrom a biological sample from a subject, wherein the A2M is present at aconcentration of at least 1.1 times higher than the concentration of A2Mpresent in the biological sample from the subject; and plasma, bonemarrow aspirate (BMA), or another body fluid from the biological sample.

One aspect provided is a method for the treatment or prophylaxis of awound in a subject, comprising administering to the wound an effectiveamount of a composition comprising a variant A2M polypeptide or portionthereof.

One aspect provided is a method for the treatment or prophylaxis of awound in a subject, comprising administering to the wound an effectiveamount of a composition comprising an agent that inhibits one or moreproteins or cells associated with formation of the FAC.

In some embodiments, the administering comprises topically applying. Insome embodiments, the administering comprises systemicallyadministering. In some embodiments, matrix metalloproteinases areinhibited in the wound. In some embodiments, FAC formation is inhibitedor FAC dissociation is promoted. In some embodiments, the wound is achronic wound. In some embodiments, the wound is a slow healing wound.In some embodiments, the wound is an incomplete healing wound. In someembodiments, the wound is an open wound. In some embodiments, the woundis closed wound. In some embodiments, the wound is characterized atleast in part by one or more of a prolonged inflammatory phase, a slowforming extracellular matrix, and a stalled or decreased rate ofepithelialization. In some embodiments, the chronic wound ischaracterized at least in part by one or more of a chronicself-perpetuating state of wound inflammation, a deficient and defectivewound extracellular matrix (ECM), poorly responding wound cells, limitedECM production, and failure of re-epithelialization due in part to lackof the necessary ECM orchestration and lack of scaffold for migration.In some embodiments, the chronic wound is characterized at least in partby one or more of prolonged inflammation and proteolytic activity,leading to ulcerative lesions prolonged fibrosis in the wound leading toscarring, progressive deposition of matrix in the affected area, longerrepair times, less wound contraction, slower re-epithelialization, andincreased thickness of granulation tissue. In some embodiments, thechronic wound is a chronic skin wound. In some embodiments, the chronicwound has not healed within one month. In some embodiments, chronicwound is selected from the group consisting of sores and ulcers. In someembodiments, the ulcers are selected from the group consisting of venousulcers, diabetic pressure ulcers, stasis ulcers, venous stasis ulcers,diabetic foot ulcers, arterial insufficiency ulcers, burn ulcers,traumatic ulcers, or any combination thereof. In some embodiments, thesores are selected from the group consisting of pressure sores. In someembodiments, the subject has undergone a cosmetic procedure. In someembodiments, the cosmetic procedure comprises a cosmetic surgery,plastic surgery, breast augmentation, hair replacement, laser skinresurfacing, tummy tuck, ear surgery, microderm-abrasion treatment, nosesurgery, spider vein treatment, eyelid surgery, thigh lift, chemicalpeel, arm lift, treatment with a dermal filler, chin surgery,liposuction, brow lift, facelift, treatment with Botulinum Toxin, skinrejuvenation procedure, implantation, tattooing, or any combinationthereof. In some embodiments, the subject is an animal. In someembodiments, the subject is a mammal. In some embodiments, the subjectis a human pig, mouse, rat, rabbit, cat, dog, monkey, frog, horse orgoat. In some embodiments, the subject is a human. In some embodiments,the composition is autologous. In some embodiments, the composition isnot immunogenic to the subject. In some embodiments, the composition isa liquid. In some embodiments, the composition comprises platelets. Insome embodiments, the composition is on a wound dressing. In someembodiments, the biological sample is a blood, BMA, or a body fluid. Insome embodiments, the composition comprises one or more additionalnon-blood derived components. In some embodiments, the one or moreadditional non-blood derived components comprise an anti-coagulant,wherein the anti-coagulant comprises EDTA, tri-sodium citrate, water forinjection (WFI), or saline. In some embodiments, the compositioncomprises one or more additional blood-derived components. In someembodiments, the one or more additional blood-derived componentscomprise platelets. In some embodiments, the composition issubstantially free of cells and particles with a diameter of at leastabout 0.1 μm, 0.2 μm, 0.6 μm or 1 μm or more. In some embodiments, thecomposition is substantially free of red blood cells. In someembodiments, the composition is substantially free of white blood cells.In some embodiments, the composition is substantially free of platelets.In some embodiments, the composition comprises a first plurality ofnon-A2M proteins and molecules. In some embodiments, the compositioncomprises a second plurality of non-A2M proteins and molecules. In someembodiments, the first plurality of non-A2M proteins and molecules arecharacterized as having a molecular weight more than about 10 kDa, andare present at a concentration of at least 1.1 times higher than foundin the biological sample from the mammal. In some embodiments, the firstplurality of non-A2M proteins and molecules are characterized as havinga molecular weight less than about 500 kDa, and are present at aconcentration of less than about 90%, 70%, 50%, 30%, or 10% of aconcentration of those proteins in the biological sample from themammal. In some embodiments, the second plurality of non-A2M proteinsand molecules are characterized as having a molecular weight more thanabout 10 kDa, and are present at a concentration of at least 1.1 timeshigher than found in the biological sample from the mammal. In someembodiments, the first plurality of non-A2M proteins and moleculescomprise cytokines; chemokines; immunomodulatory mediators, peptides,proteins, DNA, RNA, carbohydrates, small molecules; proteases;degradative proteins; or any combination thereof. In some embodiments,the cytokines comprise interleukins, tumor necrosis factors (TNFs),monocyte chemoattractant proteins (MCPs), macrophage inflammatoryproteins (MIPs), tumor growth factors (TGFs), matrix metalloproteases(MMPs), or any combination thereof. In some embodiments, the firstplurality of non-A2M proteins and molecules are characterized as havinga molecular weight of less than about 100 kDa. In some embodiments, thefirst plurality of non-A2M proteins and molecules are characterized ashaving a molecular weight of less than about 50 kDa. In someembodiments, the first plurality of non-A2M proteins and molecules arecharacterized as having a molecular weight of less than about 10 kDa. Insome embodiments, the second plurality of non-A2M proteins and moleculesare characterized as having a molecular weight more than about 50 kDa.In some embodiments, the second plurality of non-A2M proteins andmolecules are characterized as having a molecular weight of more thanabout 100 kDa. In some embodiments, the second plurality of non-A2Mproteins and molecules are characterized as having a molecular weight ofmore than about 500 kDa. In some embodiments, the first plurality ofnon-A2M proteins and molecules are characterized as having a molecularweight more than about 50 kDa. In some embodiments, the first pluralityof non-A2M proteins and molecules are characterized as having amolecular weight of more than about 100 kDa. In some embodiments, thefirst plurality of non-A2M proteins and molecules are characterized ashaving a molecular weight of more than about 500 kDa. In someembodiments, the A2M is present at a concentration of at least 1.1, 1.2,1.3, 1.4, 1.5, 2, 3, 5, 10, or 20 times higher than the concentration ofA2M present in the biological sample from the mammal. In someembodiments, protease activity is inhibited at a site of administration.In some embodiments, the subject has been previously diagnosed as havinga wound. In some embodiments, the rate of wound healing is increased inthe subject. In some embodiments, the treating results in a reduction inseverity, size, infection, or bleeding, or an increase in a rate ofprogression, of the wound. In some embodiments, the method furthercomprises administering one or more additional carriers or drugs. Insome embodiments, the one or more additional carriers or drugs steroidalor non-steroidal anti-inflammatory agents, ibuprofen, aspirin,paracetamol, glucocorticoids, acetaminophen, hydrocortisone,betamethosone, local anesthetics, antimicrobial agents, growth factors,protease inhibitors. an antiseptic, an antibiotic, cephalosporins,penicillins, tetracyclines, aminoglycosides, antifungals, sulphadiazine,chloramphenicol, erythromycin, vancomycin, trimethoprim, silver,chlorhexidine, povidone iodine, triclosan, sucralfate, quarternaryammonium salts, or any combination thereof. In some embodiments, thecomposition further comprises one or more pharmaceutical acceptableexcipients.

In some embodiments, the variant A2M polypeptide comprising a baitregion, the bait region of the variant A2M polypeptide comprising aplurality of protease recognition sites arranged in series. In someembodiments, the variant A2M polypeptide protein is a recombinantprotein. In some embodiments, the variant A2M polypeptide protein isproduced in a host comprising bacteria, yeast, fungi, insect, ormammalian cells, or a cell free system. In some embodiments, the variantA2M polypeptide protein is characterized by an enhanced nonspecificinhibition of serine proteases, threonine proteases, cysteine proteases,aspartate proteases, metalloproteases, glutamic acid proteases, or anycombination thereof. In some embodiments, the variant A2M polypeptideprotein further comprises PEG with abnormal glycosylation sites. In someembodiments, the variant A2M polypeptide protein has a longer half-lifethan the half-life of a wild type A2M protein when disposed within achronic wound of a subject. In some embodiments, the plurality ofprotease recognition sites comprise one or more protease substrate baitregions from one or more proteins other than A2M, one or more additionalprotease bait regions from A2M, one or more non-natural proteinsequences, or any combination thereof, wherein the modified A2M proteinis characterized by at least a 10% increase in protease inhibitoryeffectiveness compared to the protease inhibitory effectiveness of awild type A2M protein. In some embodiments, the non-natural proteinsequences comprise one or more protease recognition sites that canfunction as bait for proteases. In some embodiments, the one or moreprotease substrate bait regions comprise consensus sequences for serineproteases, threonine proteases, cysteine proteases, aspartate proteases,metalloproteinases, glutamic acid proteases, or any combination thereof.In some embodiments, the protease substrate bait regions comprise one ormore consensus sequences for one or more proteases from one or moreorganisms. In some embodiments, the one or more organisms comprise,animals, plants, bacteria, yeast, fish, reptiles, amphibians, or fungi.In some embodiments, one or more of the one or more protease substratebait regions from the one or more proteins other than A2M are the same.In some embodiments, one or more of the one or more protease substratebait regions from A2M are the same. In some embodiments, one or more ofthe one or more protease substrate bait regions from the one or morenon-natural protein sequences are the same. In some embodiments, one ormore of the one or more protease substrate bait regions from the one ormore proteins other than A2M or from the one or more non-natural proteinsequences comprise a suicide inhibitor; wherein the suicide inhibitor isoperable to covalently attach a protease to A2M. In some embodiments,one or more of the one or more protease substrate bait regions are fromdifferent species. In some embodiments, the variant A2M polypeptidecomprises one or more non-natural bait regions, wherein the one or morenon-natural bait regions comprise one or more protease recognition sitesnot present in a wild-type A2M polypeptide. In some embodiments, thevariant A2M polypeptide is characterized by at least a 10% enhancedinhibition of one or more proteases compared to a wild-type A2Minhibition of the one or more proteases. In some embodiments, theenhanced inhibition comprises enhanced nonspecific inhibition. In someembodiments, the enhanced inhibition comprises enhanced specificinhibition. In some embodiments, the protease comprises a serineprotease, threonine protease, cysteine protease, aspartate protease,metalloprotease, glutamic acid protease, or any combination thereof. Insome embodiments, the protease comprises MMP1 (Interstitialcollagenase), MMP2 (Gelatinase-A), MMP3 (Stromelysin 1), MMP7(Matrilysin, PUMP 1), MMP8 (Neutrophil collagenase), MMP9(Gelatinase-B), MMP10 (Stromelysin 2), MMP11), Stromelysin 3), MMP12(Macrophage metalloelastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP),MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18 (Collagenase 4,xcol4, xenopus collagenase), MMP19 (RASI-1, stromelysin-4), MMP20(Enamelysin), MMP21 (X-MMP), MMP23A (CA-MMP), MMP23B MMP24 (MT5-MMP),MMP25 (MT6-MMP), MMP26 (Matrilysin-2, endometase), MMP27 (MMP-22,C-MMP), MMP28 (Epilysin); A Disintegrin and Metalloproteinase withThrombospondin Motifs protease, such as ADAMTS1, ADAMTS2, ADAMTS3,ADAMTS4, ADAMTS5 (ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8 (METH-2),ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16,ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20; chymotrypsin; trypsin; elastase;compliment factors; clotting factors; thrombin; plasmin; subtilisin;Neprilysin; Procollagen peptidase; Thermolysin; Pregnancy-associatedplasma protein A; Bone morphogenetic protein 1; Lysostaphin; Insulindegrading enzyme; ZMPSTE2; acetylcholinesterase; or a combinationthereof. In some embodiments, the protease comprises ADAMTS4, ADAMTS 5,MMP13, or a combination thereof. In some embodiments, the modified A2Mpolypeptide is characterized by at least a 10% enhanced inhibition ofFAC formation compared to a wild-type A2M inhibition of FAC formation.In some embodiments, the one or more non-natural bait regions arederived from one or more proteins other than A2M. In some embodiments,the one or more proteins other than A2M are from a non-human organism.In some embodiments, the non-human organism comprises an animal, plant,bacterium, yeast, fish, reptile, amphibian, or fungi. In someembodiments, the one or more non-natural bait regions comprise SEQ IDNOs 5-66. In some embodiments, the variant A2M polypeptide comprises SEQID NO 4, or a fragment thereof. In some embodiments, the one or morenon-natural bait regions comprise SEQ ID NOs 5-66, or fragments thereof.In some embodiments, the wild-type A2M polypeptide comprises SEQ ID NO3, or a fragment thereof. In some embodiments, one or more of the one ormore non-natural bait regions comprise a suicide inhibitor; wherein thesuicide inhibitor is operable to covalently attach a protease to thevariant A2M polypeptide. In some embodiments, the one or more proteaserecognition sites comprise 2 or more copies of the one or more proteaserecognition sequences. In some embodiments, the one or more non-naturalbait regions comprise 2 or more copies of the one or more non-naturalbait regions. In some embodiments, the variant A2M polypeptide comprisesa wild-type A2M bait region sequence. In some embodiments, the variantA2M polypeptide is a recombinant polypeptide. In some embodiments, theone or more protease recognition sites comprise a consensus sequence fora protease. In some embodiments, the variant A2M polypeptide comprisesone or more modified glycosylation sites. In some embodiments, the oneor more modified glycosylation sites are functionalized with PEG. Insome embodiments, the variant A2M polypeptide has at least a 10% longerhalf-life than the half-life of a wild type A2M polypeptide whendisposed within the subject.

In some embodiments, the agent comprises an antibody, polypeptide,nucleotide, or small molecule. In some embodiments, the agent binds tothe FAC but not to the individual components of the complex separately.In some embodiments, the agent comprises a recombinant aggrecan G3domain, wherein the domain contains the aggrecan G3 Lectin domain andcompetitively binds to fibronectin; and wherein the newly formed complexlacks the binding site to PAMP receptor and the binding site DAMPreceptor. In some embodiments, the agent comprises a recombinantfibronectin fragment, wherein the fragment comprises a G3 binding domainand competitively binds to aggrecan, and wherein the newly formedfibronectin fragment aggrecan G3 complex lacks the binding site to PAMPreceptor, and the DAMP receptor. In some embodiments, the agentcomprises an aggrecan antibody. In some embodiments, the agent comprisesa fibronectin antibody. In some embodiments, the agent comprises anantibody that binds to the PAMP receptor recognition domain of aggrecan,the DAMP receptor recognition domain of aggrecan, or both, therebyinhibiting activation of monocytes and other cells. In some embodiments,the agent comprises an antibody that binds to the PAMP receptorrecognition domain of fibronectin, the DAMP receptor recognition domainof fibronectin, or both, thereby inhibiting activation of monocytes andother cells. In some embodiments, the agent comprises a PAMP receptor orDAMP receptor that binds to the PAMP domain of aggrecan G3, the DAMPdomain of aggrecan G3, or both, thereby inhibiting activation ofmonocytes and other cells. In some embodiments, the agent comprises asoluble form of the PAMP receptor or DAMP receptor that binds to thePAMP domain of fibronectin, the DAMP domain of fibronectin, or both,thereby inhibiting activation of monocytes and other cells. In someembodiments, the agent inhibits production of proinflammatory cytokines,chemokines, proteases, or any combination thereof. In some embodiments,the agent inhibits fibroblast cells, thereby inhibiting production offibronectin, recruitment of other fibroblast cells, or a combinationthereof. In some embodiments, the agent is identified using one or morehigh-throughput screening methods. In some embodiments, the smallmolecule or polypeptide inhibits FAC formation, inhibits activation ofmonocytes, inhibits increased production of fibronectin, inhibitsrecruitment of fibroblast cells, binds to the DAMP domain offibronectin, binds to the DAMP domain of aggrecan G3, binds to the PAMPdomain of fibronectin, or binds to the PAMP domain of aggrecan G3. Insome embodiments, the small molecule or polypeptide inhibits FACformation by competitively binding to fibronectin or aggrecan. In someembodiments, the small molecule or polypeptide binds to the FAC complexresulting in dissociation or degradation of the FAC complex. In someembodiments, inhibiting the formation of the FAC comprises inhibiting ofone or more steps in FAC formation. In some embodiments, the one or moresteps in FAC formation comprise production of fibronectin in the ECM,production of proteases and metalloproteases, production of inflammatorycytokines and chemokines, degradation of aggrecan in cartilage, andproduction of aggrecan G3 domain fragment.

In one aspect, provided herein is a system for enrichment of A2M from afluid sample comprising: a centrifuge; a flow filtration modulecomprising one or more filters; and an A2M enriched retentate from thefluid sample.

In some embodiments, the system further comprises a pump adapted to befluidly coupled to the flow filtration module, and produce a flow of thefluid sample that passes through an inlet and an outlet of the flowfiltration module. In some embodiments, the pump is fluidly coupled tothe flow filtration module upstream of the inlet. In some embodiments,the pump is fluidly coupled to the flow filtration module downstream ofthe outlet. In some embodiments, the centrifuge comprises a supernatantof the fluid sample. In some embodiments, the flow filtration modulecomprises a supernatant of the fluid sample. In some embodiments, apellet of the centrifuged fluid sample is not in the flow filtrationmodule. In some embodiments, the supernatant of the fluid sample is fromthe centrifuge. In some embodiments, red blood cells have beensubstantially removed from the supernatant. In some embodiments, whiteblood cells have been substantially removed from the supernatant. Insome embodiments, a filter of the one or more filters has a pore size ofat least about 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm. In some embodiments, afilter of the one or more filters has a pore size of at most about 10,50, 100, 200, 300, 400, or 500 kDa. In some embodiments, a filter of theone or more filters comprise a cross flow filter. In some embodiments,the flow filtration module further comprises a permeate collectionreservoir. In some embodiments, the permeate collection reservoir storesa permeate from the one or more filters. In some embodiments, aretentate of the one or more filters comprises the A2M enrichedretentate. In some embodiments, the A2M enriched retentate remains inthe permeate collection reservoir. In some embodiments, a retentate ofthe one or more filters comprises cells and particles from the fluidsample comprising a diameter of at least about 0.1 μm, 0.2 μm, 0.6 μm,or 1 μm. In some embodiments, a retentate of the one or more filterscomprises a concentration of proteins with a molecular weight of atleast about 10, 50, 100, 200, 300, 400, or 500 kDa, of at least about1.1 times their concentration in the sample. In some embodiments, apermeate from the one or more filters has a concentration of proteinshaving a molecular weight less than about 10, 50, 100, 200, 300, 400, or500 kDa of less than about 90%, 80%, 60%, 30%, or 10% of theconcentration of those proteins in the sample. In some embodiments, theone or more filters comprise two or more filters.

In one aspect, provided herein is a system for concentrating A2M from afluid sample comprising: a flow filtration module comprising an inlet,an outlet, and two or more filters fluidly connected in series betweenthe inlet and outlet; and an A2M enriched retentate from the fluidsample.

In some embodiments, the system further comprises a pump adapted to befluidly coupled to the flow filtration module, and produce a flow of thefluid sample that passes through the filter unit from the inlet to theoutlet. In some embodiments, the pump is fluidly coupled to the flowfiltration module upstream of the inlet. In some embodiments, the pumpis fluidly coupled to the flow filtration module downstream of theoutlet. In some embodiments, the system further comprises a centrifuge.In some embodiments, the two or more filters are fluidly connected inseries between an inlet and an outlet of the flow filtration module. Insome embodiments, a first of the two or more filters has a pore size ofat least about 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm, and a second of the twoor more filters has a pore size of at most about 10, 50, 100, 200, 300,400, or 500 kDa. In some embodiments, the fluid sample passes throughthe two or more filters to produce the A2M enriched retentate. In someembodiments, the two or more filters comprise a first and a secondfilter, wherein the second filter is downstream of the first filter. Insome embodiments, the first and the second of the two or more filterscomprise a first and a second cross flow filter. In some embodiments, aretentate of the second filter comprises the A2M enriched retentate. Insome embodiments, a retentate of the first filter comprises cells andparticles from the fluid sample comprising a diameter of at least about0.1 μm, 0.2 μm, 0.6 μm, or 1 μm. In some embodiments, a retentate of thesecond filter comprises proteins with a molecular weight of at leastabout 10, 50, 100, 200, 300, 400, or 500 kDa, wherein the concentrationof those proteins is at least about 1.1 times their concentration in thefluid sample. In some embodiments, the permeate from the second filterhas a concentration of proteins having a molecular weight less thanabout 10, 50, 100, 200, 300, 400, or 500 kDa of less than about 90%,80%, 60%, 30%, or 10% of the concentration of those proteins in thesample. In some embodiments, a first permeate from the first filterflows through the second filter. In some embodiments, the flowfiltration module further comprises a first and a second permeatecollection reservoir. In some embodiments, the first permeate collectionreservoir stores a permeate from the first filter and a retentate of thesecond filter. In some embodiments, the second permeate collectionreservoir stores a permeate from the second filter. In some embodiments,the A2M enriched retentate remains in the first permeate collectionreservoir. In some embodiments, the flow filtration module is a dead endand/or tangential flow filtration module. In some embodiments, the fluidsample comprises a biological sample. In some embodiments, thebiological sample is from an animal. In some embodiments, the biologicalsample is from a mammal. In some embodiments, the biological sample isfrom a human. In some embodiments, the system further comprises one ormore waste modules in fluid connection with the flow filtration module.In some embodiments, the one or more waste modules are downstream of theflow filtration module. In some embodiments, the one or more wastemodules comprise particles, and other molecules with a diameter of atleast about 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm from the fluid sample. Insome embodiments, the one or more waste modules comprise proteins with amolecular weight of less than about 10, 50, 100, 200, 300, 400, or 500kDa from the fluid sample. In some embodiments, the fluid sample isflowing through the one or more filters in sequence using thecentrifuge, a pump, or a combination thereof. In some embodiments, thefluid sample is flowing through the two or more filters in sequenceusing the centrifuge, a pump, or a combination thereof. In someembodiments, the system further comprises a collection module in fluidconnection with the flow filtration module. In some embodiments, thecollection module is downstream of the centrifuge. In some embodiments,the collection module comprises the A2M enriched retentate. In someembodiments, the system further comprises a sample loading moduleoperable to introduce the fluid sample into the system. In someembodiments, the sample loading module is directly or indirectlyattached to the blood stream of a subject. In some embodiments, the A2Menriched retentate comprises A2M at a concentration of at least 1.1times higher than found in the fluid sample. In some embodiments, theA2M enriched retentate comprises a first plurality of non-A2M proteinsand molecules. In some embodiments, the A2M enriched retentate comprisesa second plurality of non-A2M proteins and molecules. In someembodiments, the first plurality of non-A2M proteins and molecules arecharacterized as having a molecular weight more than about 10, 50, 100,300, or 500 kDa, and are present at a concentration of at least 1.1times higher than found in the fluid sample. In some embodiments, thefirst plurality of non-A2M proteins and molecules are characterized ashaving a molecular weight less than about 500 kDa, and are present at aconcentration of less than about 90%, 70%, 50%, 30%, or 10% of aconcentration of those proteins in the fluid sample. In someembodiments, the second plurality of non-A2M proteins and molecules arecharacterized as having a molecular weight more than about 10 kDa, andare present at a concentration of at least 1.1 times higher than foundin the fluid sample. In some embodiments, the first plurality of non-A2Mproteins and molecules comprise cytokines; chemokines; immunomodulatorymediators, peptides, proteins, DNA, RNA, carbohydrates, small molecules;proteases; degradative proteins; or any combination thereof. In someembodiments, the cytokines comprise interleukins, tumor necrosis factors(TNFs), monocyte chemoattractant proteins (MCPs), macrophageinflammatory proteins (MIPs), tumor growth factors (TGFs), matrixmetalloproteases (MMPs), or any combination thereof. In someembodiments, red blood cells have been substantially removed from thefluid sample. In some embodiments, white blood cells have beensubstantially removed from the fluid sample. In some embodiments,platelets have been substantially removed from the fluid sample. In someembodiments, red blood cells have been substantially removed from theA2M enriched retentate. In some embodiments, white blood cells have beensubstantially removed from the A2M enriched retentate. In someembodiments, platelets have been substantially removed from the A2Menriched retentate. In some embodiments, the A2M enriched retentatecomprises platelets. In some embodiments, the A2M enriched retentate isobtained in less than about 15 minutes, 30 minutes, 45 minutes, 1 hour,or 3 hours.

In one aspect, provided herein is a method for enrichment of A2M from abiological sample obtained from an animal comprising: flowing thebiological sample through one or more filters of a flow filtrationmodule, thereby separating the sample into a permeate and a retentate;and collecting the retentate; wherein the collected retentate is an A2Menriched retentate, wherein the concentration of A2M in the A2M enrichedretentate is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, or 20times higher than the concentration of A2M in the sample, and whereinthe concentration in the A2M enriched retentate of proteins having amolecular weight less than about 10, 50, 100, 200, 300, 400, or 500 kDais less than 90%, 80%, 60%, 30%, or 10% of the concentration of thoseproteins in the sample.

In some embodiments, the method further comprises removing cells andparticles with a diameter of at least about 0.1 μm, 0.2 μm, 0.6 μm, or 1μm from the sample before (a). In some embodiments, the removingcomprises centrifuging the biological sample, and obtaining asupernatant of the fluid sample. In some embodiments, the removingcomprises flowing the biological sample through a filter. In someembodiments, the flowing comprises flowing the biological sample througha first filter of the one or more filters, thereby separating thebiological sample into a first permeate and a first retentate, andflowing the first permeate through a second filter of the one or morefilters, thereby separating the biological sample into a second permeateand a second retentate; wherein the second retentate is the A2M enrichedretentate.

In one aspect, provided herein is a method of enriching A2M from abiological sample from an animal comprising: removing cells andparticles with a diameter of at least about 0.1 μm, 0.2 μm, 0.6 μm, or 1μm from a biological sample to produce a fluid sample, and flowing thefluid sample through one or more filters of a flow filtration module,thereby producing a an A2M enriched retentate.

In some embodiments, the removing comprises centrifuging the biologicalsample, and obtaining a supernatant of the fluid sample. In someembodiments, the removing comprises flowing the biological samplethrough a filter. In some embodiments, the method further comprisesdiscarding or retaining a pellet of the fluid sample. In someembodiments, the flowing comprises flowing the fluid sample through afirst filter of the one or more filters, thereby separating the fluidsample into a first permeate and a first retentate, and flowing thefirst permeate through a second filter of the one or more filters,thereby separating the fluid sample into a second permeate and a secondretentate; wherein the second retentate is the A2M enriched retentate.In some embodiments, a first filter of the one or more filters has apore size of at least about 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm. In someembodiments, a first filter of the one or more filters has a pore sizeof at least about 10, 50, 100, 200, 300, 400, or 500 kDa. In someembodiments, a retentate from the one or more filters has aconcentration of proteins having a molecular weight less than about 10,50, 100, 200, 300, 400, or 500 kDa of less than about 99%, 95%, 90%,80%, 60%, 30%, or 10% of the concentration of those proteins in thebiological sample. In some embodiments, a permeate from the one or morefilters has a concentration of proteins having a molecular weight lessthan about 10, 50, 100, 200, 300, 400, or 500 kDa of more than about99%, 95%, 90%, 80%, 60%, 30%, or 10% of the concentration of thoseproteins in the biological sample. In some embodiments, the one or morefilters comprise two or more filters.

In one aspect, provided herein is a method for enriching A2M from abiological sample obtained from an animal comprising: flowing thebiological sample through two or more filters of a flow filtrationmodule, thereby separating the biological sample into two or morepermeates and two or more retentates; and collecting at least one of thetwo or more retentates; wherein the collected retentate is an A2Menriched retentate.

In some embodiments, the flowing comprises flowing the biological samplethrough a first filter of the two or more filters, thereby separatingthe sample into a first permeate and a first retentate, and flowing thefirst permeate through a second filter of the two or more filters,thereby separating the sample into a second permeate and a secondretentate; wherein the second retentate is the collected retentate. Insome embodiments, a first filter of the two or more filters has a poresize of at least about 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm. In someembodiments, a first filter of the two or more filters has a pore sizeof at least about 10, 50, 100, 200, 300, 400, or 500 kDa. In someembodiments, a second filter of the two or more filters has a pore sizeof at least about 10, 50, 100, 200, 300, 400, or 500 kDa. In someembodiments, the first filter comprises a first cross-flow filter. Insome embodiments, the second filter comprises a second cross-flowfilter. In some embodiments, the method further comprises filtering apermeate of the first filter with the second filter. In someembodiments, the method further comprises collecting a retentate of thesecond filter. In some embodiments, the retentate of the second filtercomprises the A2M enriched retentate. In some embodiments, the methodfurther comprises storing the A2M enriched retentate in a second filterretentate reservoir in fluid connection with the second filter. In someembodiments, the method further comprises storing a retentate of thefirst filter in a reservoir in fluid connection with the first filter.In some embodiments, the two or more filters are fluidly connected inseries between an inlet and an outlet of the flow filtration module. Insome embodiments, the fluid sample passes through the two or morefilters to produce the A2M enriched retentate. In some embodiments, thetwo or more filters comprise a first and a second filter, wherein thesecond filter is downstream of the first filter. In some embodiments,the first and the second of the two or more filters comprise a first anda second cross flow filter. In some embodiments, a retentate of thesecond filter comprises the A2M enriched retentate. In some embodiments,a retentate of the first filter comprises cells and particles from thefluid sample comprising a diameter of at least about 0.1 μm, 0.2 μm, 0.6μm, or 1 μm. In some embodiments, in a retentate of the second filterthe concentration of proteins with a molecular weight of at least about10, 50, 100, 200, 300, 400, or 500 kDa is at least about 1.1 times theirconcentration in the biological sample. In some embodiments, a permeatefrom the second filter has a concentration of proteins having amolecular weight less than about 10, 50, 100, 200, 300, 400, or 500 kDaof more than about 99%, 95%, 90%, 80%, 60%, 30%, or 10% of theconcentration of those proteins in the s biological ample. In someembodiments, a first permeate from the first filter flows through thesecond filter. In some embodiments, the flow filtration module furthercomprises a first and a second permeate collection reservoir. In someembodiments, the first permeate collection reservoir stores a permeatefrom the first filter and a retentate of the second filter. In someembodiments, the second permeate collection reservoir stores a permeatefrom the second filter. In some embodiments, the A2M enriched retentateremains in the first permeate collection reservoir. In some embodiments,the flow filtration module comprises and inlet and an outlet, andwherein the one or more filters are fluidly connected in series betweenthe inlet and the outlet. In some embodiments, the flow filtrationmodule comprises and inlet and an outlet, and wherein the two or morefilters are fluidly connected in series between the inlet and theoutlet. In some embodiments, the flowing comprises pumping with a pump,wherein the pump is fluidly connected to the flow filtration moduleupstream of the inlet or downstream of the outlet. In some embodiments,the pumping comprises manually actuating the pump. In some embodiments,the biological sample comprises plasma. In some embodiments, the methodfurther comprises removing red blood cells from the biological sample.In some embodiments, the method further comprises removing white bloodcells from the biological sample. In some embodiments, the methodfurther comprises removing platelets from the biological sample. In someembodiments, the red blood cells, white blood cells, platelets, or anycombination thereof, are removed by flowing or passing the samplethrough the filters. In some embodiments, the method further comprisesadding one or more blood derived components to the biological sample orthe A2M enriched retentate. In some embodiments, the one or more bloodderived components comprise platelets. In some embodiments, a filter ofthe one or more filters is a hollow fiber tangential flow filter. Insome embodiments, a filter of the two or more filters is a hollow fibertangential flow filter. In some embodiments, a filter of the one or morefilters comprises a charge, immobilized molecules, or a combinationthereof, thereby enhancing the selectivity of the filter. In someembodiments, a filter of the two or more filters comprises a charge,immobilized molecules, or a combination thereof, thereby enhancing theselectivity of the filter. In some embodiments, the immobilizedmolecules comprise antibodies, proteins, receptors, ligands,carbohydrates, nucleotides, RNA, DNA, or any combination thereof. Insome embodiments, enhancing the selectivity comprises enhancing theability to retain A2M, enhancing the ability to not retain moleculesthat are not A2M, or a combination thereof. In some embodiments, theflowing comprises applying tangential force filtration, one or morecentrifugation steps, gravitational forces, mechanical forces, or anycombination thereof. In some embodiments, the mechanical force comprisesa pump, centrifugal force, or gas pressure, or any combination thereof.In some embodiments, the method further comprises adding one or morenon-blood derived components, one or more blood derived components, or acombination thereof, to the biological sample or the A2M enrichedretentate. In some embodiments, the one or more additional non-bloodderived components comprises an anti-coagulant, preservative, excipient,diluent, or other additive. In some embodiments, the anti-coagulantcomprises EDTA, tri-sodium citrate, water for injection (WFI), saline,or ACD-A. In some embodiments, the diluent is a WFI solution or a salinesolution. In some embodiments, the one or more additional blood derivedcomponents comprise platelets. In some embodiments, the biologicalsample is a mammalian sample. In some embodiments, the biological sampleis a human sample. In some embodiments, the biological sample iscollected with the aid of an additional absorbent, adsorbent, orcapillary materials or devices selected from the group of needle-syringecombo, sponges, wicks, pledgets, sutures, hydrophilic catheters,hydrophobic catheters, hollow-lumen catheters, or any combinationthereof. In some embodiments, the A2M enriched retentate is obtained inless than about 15 minutes, 30 minutes, 45 minutes, 1 hour, or 3 hours.In some embodiments, the concentration of A2M in the A2M enrichedretentate is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, or 20times higher than the concentration of A2M in the sample. In someembodiments, the concentration in the A2M enriched retentate of proteinshaving a molecular weight less than about 10, 50, 100, 200, 300, 400, or500 kDa is less than 90%, 80%, 60%, 30%, or 10% of the concentration ofthose proteins in the sample. In some embodiments, the flow filtrationmodule is a dead end and/or tangential flow filtration module.

In one aspect, provided herein is a system for manual flow of abiological sample comprising: a first retentate chamber comprising aflow path comprising a first end with a first inlet and second end witha second inlet; a first permeate chamber comprising a first outlet andoptionally a second outlet; a first semi-permeable membrane orientedbetween the first retentate chamber and the first permeate chamberhaving an average pore size of at least about 10, 50, 100, 200, 300,400, or 500 kDa that permits liquid flow from the first retentatechamber to the first permeate chamber; and a first injector comprising amanual actuator in fluid connection with the first end, and a secondinjector comprising a manual actuator in fluid connection with thesecond end.

In some embodiments, the system further comprises a second retentatechamber comprising a flow path comprising a third end with a third inletand fourth end with a fourth inlet; a second permeate chamber comprisinga second outlet, wherein the second outlet is optionally in fluidconnection with the first retentate chamber; a second semi-permeablemembrane oriented between the second retentate chamber and the secondpermeate chamber having an average pore size of at least about 0.1 μm,0.2 μm, 0.6 μm, or 1 μm that permits liquid flow from the secondretentate chamber to the second permeate chamber; and a third injectorcomprising a manual actuator in fluid connection with the third end, anda fourth injector comprising a manual actuator in fluid connection withthe fourth end.

In some embodiments, the first injector is connected to the first inletvia an adapter. In some embodiments, the system further comprises afirst vessel connected to the first inlet via the adapter. In someembodiments, the first vessel comprises sterile air. In someembodiments, the first vessel comprises a syringe. In some embodiments,the first vessel is disposed below the first membrane. In someembodiments, the first vessel is disposed above the first membrane. Insome embodiments, the first vessel is oriented perpendicular to asurface of the first membrane. In some embodiments, the second injectoris connected to the second inlet via an adapter. In some embodiments,the first outlet is connected to a first vessel. In some embodiments,the first vessel comprises a syringe. In some embodiments, the firstvessel comprises a permeate of the biological sample. In someembodiments, the adapter is a valve. In some embodiments, the valve is astopcock. In some embodiments, the first inlet, second inlet, thirdinlet, fourth inlet, first outlet, second outlet, or any combinationthereof, comprises an adapter. In some embodiments, the adapter is aLuer-lock adapter, a Hose-Barb adapter, or a sanitary tri-cloveradaptor. In some embodiments, the Luer-lock adapter is closed, capped,or sealed. In some embodiments, the first inlet, second inlet, thirdinlet, fourth inlet, first outlet, second outlet, or any combinationthereof, is closed, capped, or sealed. In some embodiments, the thirdinjector is connected to the third inlet via an adapter. In someembodiments, the system further comprises a second vessel connected tothe third inlet via the adapter. In some embodiments, the second vesselcomprises sterile air. In some embodiments, the second vessel comprisesa syringe. In some embodiments, the second vessel is disposed below thesecond membrane. In some embodiments, the second vessel is disposedabove the second membrane. In some embodiments, the second vessel isoriented perpendicular to a surface of the second membrane. In someembodiments, the fourth injector is connected to the fourth inlet via anadapter. In some embodiments, the second outlet is in fluid connectionwith the first retentate chamber. In some embodiments, the second outletis in fluid communication with a second vessel. In some embodiments, theconnection is via a second outlet flow path. In some embodiments, thesecond vessel comprises a container or bag. In some embodiments, thesecond vessel comprises a permeate of the biological sample. In someembodiments, the permeate of the biological sample does not comprisecells. In some embodiments, the adapter is a valve. In some embodiments,the valve is a stopcock. In some embodiments, the second outlet flowpath is in fluid communication with the first retentate chamber via aconnecting flow path. In some embodiments, the second outlet flow pathis connected to the connecting flow path via a valve. In someembodiments, the valve allows fluid to flow from the second outlet flowpath to the second vessel in a first orientation, and wherein the valveallows fluid to flow from the second vessel to the first retentatechamber in a second orientation. In some embodiments, the second outletis in fluid connection with a collection vessel. In some embodiments,the collection vessel is adapted to fit the first or second inlet. Insome embodiments, the collection vessel is adapted to fit an adapterconnected to the first or second inlet. In some embodiments, thecollection vessel is the first or second injector. In some embodiments,the system further comprises an A2M enriched retentate contained in thefirst injector or the second injector, the A2M enriched retentatecomprising: A2M isolated from a biological sample from an animal,wherein the A2M is present at a concentration of at least 1.1 timeshigher than the concentration of A2M present in the biological samplefrom the animal; and plasma, bone marrow aspirate (BMA), or another bodyfluid from the biological sample. In some embodiments, the A2M enrichedretentate comprises proteins with a molecular weight of at least about10, 50, 100, 200, 300, 400, or 500 kDa present at a concentration of atleast 1.1 times higher than found in the biological sample from theanimal. In some embodiments, the A2M enriched retentate comprises aconcentration of molecules with a molecular weight less than 10, 50,100, 200, 300, 400, or 500 kDa is less than 90%, 70%, 50%, 30%, or 10%of the concentration of those proteins and/or fold concentration of A2Mpresent in the biological sample from the animal. In some embodiments,the biological sample is a blood sample, BMA, or other body fluid. Insome embodiments, the A2M enriched retentate is substantially free ofcells and particles with a diameter of at least about 0.1 μm, 0.2 μm,0.6 μm, or 1 μm, and comprises a reduced concentration of proteins andother molecules with a molecular weight of at least about 10, 50, 100,200, 300, 400, or 500 kDa compared to the biological sample. In someembodiments, the A2M enriched retentate is for autologous delivery intoor onto one or more wounds of the animal. In some embodiments, the A2Menriched retentate is an autologous composition. In some embodiments,the first inlet is disposed to introduce a biological sample into thefirst retentate chamber and parallel to the surface of the firstmembrane. In some embodiments, the second inlet is disposed to introducea biological sample into the first retentate chamber and parallel to thesurface of the first membrane. In some embodiments, the third and/orfourth inlet is disposed to introduce a biological sample into thesecond retentate chamber and parallel to the surface of the secondmembrane. In some embodiments, the system further comprises a means forproviding the biological sample or permeate of the biological sample tothe first or second inlet of the first retentate chamber, and a meansfor controlling a filtration rate of the biological sample through thefirst membrane and into the first permeate chamber. In some embodiments,the system further comprises a means for providing a the biologicalsample to the third or fourth inlet of the second retentate chamber, anda means for controlling a filtration rate of the biological samplethrough the second membrane and into the second permeate chamber. Insome embodiments, the first retentate chamber is cylindrical, andwherein the first outlet is disposed perpendicular to a surface of thefirst and/or second membrane. In some embodiments, the second retentatechamber is cylindrical, and wherein the second outlet is disposedperpendicular to a surface of the second and/or first membrane. In someembodiments, the second retentate chamber is disposed above the firstretentate chamber. In some embodiments, the second retentate chamber isparallel to the first retentate chamber. In some embodiments, the firstinlet and/or the second inlet is disposed above the first membrane. Insome embodiments, the third inlet and/or the fourth inlet is disposedabove the second and/or first membrane. In some embodiments, the firstinlet and/or the second inlet is oriented perpendicular to a surface ofthe first membrane. In some embodiments, the third inlet and/or thefourth inlet is oriented perpendicular to a surface of the second and/orfirst membrane. In some embodiments, the first inlet and/or the secondinlet is oriented parallel to a surface of the first membrane. In someembodiments, the third inlet and/or the fourth inlet is orientedparallel to a surface of the second and/or first membrane. In someembodiments, the system is portable.

In one aspect, provided herein is a method for enrichment of A2M from abiological sample obtained from an animal comprising: flowing abiological sample into a system comprising: a first retentate chambercomprising a flow path comprising a first end with a first inlet andsecond end with a second inlet; a first permeate chamber comprising afirst outlet; a first semi-permeable membrane oriented between the firstretentate chamber and the first permeate chamber that permits liquidflow from the first retentate chamber to the first permeate chamber; anda first injector comprising a manual actuator in fluid connection withthe first end, and a second injector comprising a manual actuator influid connection with the second end; through the first inlet, over thefirst membrane of the first retentate chamber, and through the secondinlet, thereby separating the sample into a permeate and a retentate;and collecting the retentate in the first or second injector, whereinthe retentate is enriched for A2M.

In some embodiments, the system of the method further comprises: asecond retentate chamber comprising a flow path comprising a third endwith a third inlet and fourth end with a fourth inlet; a second permeatechamber comprising a second outlet, wherein the second outlet isoptionally in fluid connection with the first retentate chamber; asecond semi-permeable membrane oriented between the second retentatechamber and the second permeate chamber that permits liquid flow fromthe second retentate chamber to the second permeate chamber; and a thirdinjector comprising a manual actuator in fluid connection with the thirdend, and a fourth injector comprising a manual actuator in fluidconnection with the fourth end; and wherein the method furthercomprises: flowing the sample through the third inlet, over the secondmembrane of the second retentate chamber, and through the fourth inlet,thereby retaining cells and other particles with an average diameter ofat least about 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm in the second retentatechamber; and flowing the permeate from the second membrane into thefirst retentate chamber.

In some embodiments, the concentration of A2M in the retentate is atleast 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, or 20 times higher thanthe concentration of A2M in the sample. In some embodiments, theretentate comprises a concentration of proteins with a molecular weightless than about 10, 50, 100, 300, or 500 kDa of less than 90%, 80%, 60%,30%, or 10% the concentration of those proteins in the biologicalsample. In some embodiments, the flowing comprises flowing the permeatefrom the second membrane through a channel connecting the second outletwith the first retentate chamber. In some embodiments, the flowingcomprises collecting the permeate from the second membrane in acollection vessel and flowing the collected permeate from the secondmembrane through the first or second inlet into the first retentatechamber. In some embodiments, the collection vessel is adapted to fitthe first or second inlet. In some embodiments, the collection vessel isadapted to fit an adapter connected to the first or second inlet. Insome embodiments, the collection vessel is the first or second injector.In some embodiments, the flowing comprises actuating the first injector.In some embodiments, the flowing further comprises actuating the secondinjector. In some embodiments, the first and second injectors areactuated on or more times. In some embodiments, the first and secondinjectors are actuated in sequence. In some embodiments, the biologicalsample comprises plasma. In some embodiments, red blood cells and whiteblood cells have been removed from the biological sample. In someembodiments, the biological sample or A2M enriched retentate furthercomprises one or more blood derived components. In some embodiments, theone or more blood derived components comprise platelets. In someembodiments, the second membrane is characterized by having a pore sizeof at least 0.1 μm, 0.2 μm, 0.6 μm, or 1 μm, or higher. In someembodiments, the first membrane comprises a hollow fiber tangential flowfilter. In some embodiments, the first membrane has a molecular weightcut-off of at most 10, 50, 100, 200, 300, 400, or 500 kDa. In someembodiments, the first and/or second membranes comprise a charge,immobilized molecules, or a combination thereof, thereby enhancing theselectivity of the one or more filters. In some embodiments, theimmobilized molecules comprise antibodies, proteins, receptors, ligands,carbohydrates, nucleotides, RNA, or DNA. In some embodiments, enhancingthe selectivity of the one or more filters comprises enhancing theability of the one or more filters to retain A2M, enhancing the abilityof the one or more filters to not retain molecules that are not A2M, ora combination thereof. In some embodiments, the flowing comprisesactuating on or more of the injectors, applying tangential forcefiltration, one or more centrifugation steps, gravitational forces,mechanical forces, or any combination thereof. In some embodiments, themechanical force comprises a manual force, a pump, centrifugal force,gas pressure, or a combination thereof. In some embodiments, the methodfurther comprises adding one or more non-blood derived components, oneor more blood derived components, or a combination thereof, to thebiological sample or A2M enriched retentate. In some embodiments, theone or more additional non-blood derived components comprises ananti-coagulant, preservative, excipient, diluent, or other additive. Insome embodiments, the anti-coagulant comprises EDTA, tri-sodium citrate,water for injection (WFI), saline, or ACD-A. In some embodiments, thediluent is a WFI solution or a saline solution. In some embodiments, theone or more additional blood derived components comprise platelets. Insome embodiments, the biological sample is from a human subject. In someembodiments, the human subject has a disease or condition treatable withthe retentate. In some embodiments, the disease or condition is a wound.In some embodiments, the wound is a chronic wound. In some embodiments,the biological sample is collected with the aid of an additionalabsorbent, adsorbent, or capillary materials or systems selected fromthe group of needle-syringe combo, sponges, wicks, pledgets, sutures,hydrophilic catheters, hydrophobic catheters, hollow-lumen catheters, orany combination thereof. In some embodiments, the method furthercomprises centrifuging the sample to remove cells and particles.

In one aspect, provided herein is a method of manufacture of amedicament comprising bringing together an amount of a compositioncomprising A2M isolated from a biological sample from an animal, whereinthe A2M is present at a concentration of at least 1.1 times higher thanthe concentration of A2M present in the biological sample from theanimal, and plasma, BMA, or another body fluid from the biologicalsample; with a pharmaceutically acceptable carrier, wherein themedicament is effective to promote wound healing.

In one aspect, provided herein is an article of manufacture comprisingpackage material containing a therapeutically effective amount of acomposition comprising A2M isolated from a biological sample from ananimal, wherein the A2M is present at a concentration of at least 1.1times higher than the concentration of A2M present in the biologicalsample from the animal, and plasma, BMA, or another body fluid from thebiological sample; together with instructions for use in the treatmentof a wound in or on a subject.

In one aspect, provided herein is a method of manufacture of amedicament comprising bringing together an amount of a compositioncomprising a variant A2M polypeptide or portion thereof effective topromote wound healing and a pharmaceutically acceptable carrier.

In one aspect, provided herein is an article of manufacture comprisingpackage material containing therapeutically effective amounts of acomposition comprising a variant A2M polypeptide or portion thereoftogether with instructions for use in the treatment of a wound in or ona subject

In one aspect, provided herein is a method of manufacture of amedicament comprising bringing together an amount a compositioncomprising an agent that inhibits one or more proteins or cellsassociated with formation of the FAC effective to promote wound healingand a pharmaceutically acceptable carrier.

In one aspect, provided herein is an article of manufacture comprisingpackage material containing therapeutically effective amounts of acomposition comprising an agent that inhibits one or more proteins orcells associated with formation of the FAC together with instructionsfor use in the treatment of a wound in or on a subject.

In some embodiments, the biological sample comprises a first biologicalsample from a first subject and a second biological sample from a secondsubject. In some embodiments, the first and second biological samplesare blood samples or plasma samples. In some embodiments, the first andsecond biological samples do not substantially comprise blood cells. Insome embodiments, the animal is a first animal and wherein the methodfurther comprises combining the A2M enriched retentate with another A2Mretentate obtained from another biological sample to form a pooledsample, wherein the another biological sample is from a second animalthat is different from the first animal. In some embodiments, thebiological samples are blood samples or plasma samples. In someembodiments, the A2M enriched retentates do not substantially compriseblood cells. In some embodiments, the method further comprises treatinga subject in need thereof with the pooled sample. In some embodiments,the pooled sample is not immunogenic to the subject. In someembodiments, the fluid sample is a biological sample, wherein thebiological sample comprises a first biological sample from a firstsubject and a second biological sample from a second subject. In someembodiments, the biological sample comprises a first biological samplefrom a first subject and a second biological sample from a secondsubject. In some embodiments, the first and second biological samplesare blood samples or plasma samples. In some embodiments, the first andsecond biological samples do not substantially comprise blood cells. Insome embodiments, the biological sample comprises a first biologicalsample from a first subject and a second biological sample from a secondsubject. In some embodiments, the first and second biological samplesare blood samples or plasma samples. In some embodiments, the first andsecond biological samples do not substantially comprise blood cells. Insome embodiments, the animal is a first animal and wherein the articlefurther comprises an A2M enriched retentate obtained from anotherbiological sample to form a pooled sample, wherein the anotherbiological sample is from a second animal that is different from thefirst animal. In some embodiments, the biological samples are bloodsamples or plasma samples. In some embodiments, the article does notsubstantially comprise blood cells.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification, including related International Application No.PCT/US2013/027159, are herein incorporated by reference to the sameextent as if each individual publication, patent, or patent applicationwas specifically and individually indicated to be incorporated byreference. In the event of a conflict between a term herein and a termincorporated by reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features are set forth with particularity in the appendedclaims. A better understanding of the features and advantages will beobtained by reference to the following detailed description that setsforth illustrative embodiments, in which the principles of devices,methods, and compositions are utilized, and the accompanying drawings ofwhich:

FIG. 1 depicts a schematic of an exemplary system. comprising onemembrane

FIG. 2 depicts a schematic of an exemplary system comprising onemembrane.

FIG. 3 depicts a schematic of an exemplary system comprising onemembrane.

FIG. 4 depicts a schematic of an exemplary system comprising onemembrane.

FIG. 5 depicts a schematic of an exemplary system comprising onemembrane.

FIG. 6 depicts a schematic of an exemplary system comprising onemembrane.

FIG. 7 depicts a schematic of an exemplary system comprising twomembranes.

FIG. 8 depicts a schematic of an exemplary system comprising twomembranes.

FIGS. 9A-9G depict the flow of a sample through an exemplary systemcomprising two membranes.

FIG. 10 depicts a graph comparing recovery of various components from asample using an automatic system, a manual system, and a systemcomprising a hemaconcentrator.

FIG. 11 depicts a graph comparing recovery of various components from asample using an automatic system, a manual system, and a systemcomprising a hemaconcentrator.

FIG. 12 depicts a schematic of view of a filtration module of a system.

FIG. 13 depicts a bar graph of the % of the indicated cells compared towhole blood (WB)) using the indicated methods.

FIG. 14 depicts a bar graph of the concentration of A2M (mg/mL) inblood, plasma, and concentrated compositions produced using theindicated methods.

FIGS. 15A-C depict bar graphs of the concentration of the indicatedanalytes in plasma and concentrated compositions produced using theindicated methods.

FIG. 16A depicts a diagram of an exemplary APIC-HL system.

FIG. 16B depicts a diagram of an exemplary APIC-HH system.

FIG. 17 depicts a flow chart of the steps for exemplary methods ofpreparing therapeutic compositions described herein.

FIG. 18 depicts a flow chart of the steps for exemplary methods ofpreparing therapeutic compositions described herein.

FIG. 19 depicts a flow chart of the steps for exemplary methods ofpreparing therapeutic compositions described herein.

FIG. 20A depicts a bar graph of the percent recovery of platelets fromwhole blood in concentrated products produced using the indicatedmethod.

FIG. 20B depicts a bar graph of the fold change of platelets inconcentrated products produced using the indicated method compared towhole blood.

FIG. 21A depicts a bar graph of the fold change of white blood cells inconcentrated products produced using the indicated method compared towhole.

FIG. 21B depicts a bar graph of the fold change of red blood cells inconcentrated products produced using the indicated method compared towhole blood.

FIG. 22 depicts a bar graph of the fold change of A2M in concentratedproducts produced using the indicated method compared to whole blood.

FIG. 23 depicts a bar graph of the fold change of platelets inconcentrated products produced using the indicated method compared towhole blood

FIG. 24 depicts a schematic of the steps and signaling pathwaysassociated with formation of a fibronectin-aggrecan complex (FAC) andthe FAC-induced activation of Damage-Associated-Molecular Pattern (DAMP)receptor signaling in cells. The combination of the two processescreates a cyclic process that continually degrades cartilage.

FIG. 25 depicts a flow chart of the steps for construct or proteinexpression.

FIG. 26 depicts the A2M structure and various domains of A2M.

FIG. 27 depicts the process of tangential flow filtration using hollowfiber filters.

FIG. 28 depicts a schematic of a system as described herein.

FIG. 29 a picture of the concentration kit/tray of a system describedherein showing one filter, a concentration bag and the filtrate bag.

FIG. 30 depicts a schematic of the components of a concentration bag ofa system described herein.

FIG. 31 depicts the components of a system described herein showing acentrifuge and a one filter system.

FIG. 32 depicts two different types of custom centrifuge tubes that canbe used in the systems described herein.

FIG. 33 depicts a schematic of a cell free concentration system asdescribed herein with concentration component utilizing two filters.

FIG. 34 depicts a graph of collagenase digestion of the indicatedsamples from a wound at the indicated serial dilutions. The samplesdepicted refer to the following: 140731NL—wound fluid (WF).140731AW—debridement tissue (DT), 140731DMB-DT, and MMP-13—matrixmetalloprotease-13. Chronic wound fluid had elevated protease activity(EPA). Debridement tissue contained relatively less collagenaseactivity.

FIG. 35 depicts a graph of collagenase activity in the indicated sampleswith and without treatment of A2M at the indicated volumes (top) andcorresponding table (bottom). Serial dilutions of Autologous PlateletIntegrated Concentrate (APIC-PRP) (140224PD, 5.5 mg/ml of A2M) werechallenged to inhibit WF digestion of FITC-collagen. 1× means equalvolumes of WF and APIC-PRP. A 1× concentration of APIC was able toinhibit 75% of the collagenases in the first 3 minutes of the digestion.3×APIC to wound fluid and collagenase activity was reduced 10-fold. Nodigestion of collagen was seen in APIC-only controls. A 1:1 mixture ofAPIC and EPA wound fluid is in the linear range of inhibition. Animproperly made APIC, with less A2M concentration, may perform worse asan inhibitor.

FIGS. 36A and B depict graphs of collagenase activity in debridementtissues (A—140731DMB-DT; B—140731AW-DT) with and without A2M treatment.Serial dilutions of APIC-PRP were challenged to inhibit DT digestion ofFITC-collagen. 1× means equal volumes of DT and APIC-PRP. A 1×concentration of APIC was able to inhibit 75% of the collagenases in thefirst 3 minutes of the digestion. Collagenase activity was reduced10-fold when 3×APIC was added to wound fluid. No digestion of collagenwas seen in APIC-only controls. Less protease activity in DT (comparedto WF) was potently inhibited.

FIGS. 37A and B depict graphs (A—bar graph; B—line graph) of collagenaseactivity in the indicated samples with and without A2M treatment at theindicated amounts. 140731NL-WF (1/400) was used to digest FITC-collagenType I in the presence or absence of a serial dilution of purifiedplasma A2M. Note that WF has numerous proteases that are inhibited byA2M, but only the collagenases would be visible in this experiment.Thus, A2M efficacy is underrepresented.

DETAILED DESCRIPTION OF THE DISCLOSURE

The details of one or more inventive embodiments are set forth in theaccompanying drawings, the claims, and in the description herein. Otherfeatures, objects, and advantages of inventive embodiments disclosed andcontemplated herein will be apparent from the description and drawings,and from the claims. As used herein, unless otherwise indicated, thearticle “a” means one or more unless explicitly otherwise provided for.As used herein, unless otherwise indicated, terms such as “contain,”“containing,” “include,” “including,” and the like mean “comprising.” Asused herein, unless otherwise indicated, the term “or” can beconjunctive or disjunctive. As used herein, unless otherwise indicated,any embodiment can be combined with any other embodiment. As usedherein, unless otherwise indicated, some inventive embodiments hereincontemplate numerical ranges. When ranges are present, the rangesinclude the range endpoints. Additionally, every sub-range and valuewithin the range is present as if explicitly written out.

DEFINITIONS

The term “substantially non-immunogenic” or “substantiallynon-antigenic” means that the composition being administered to asubject does not elicit an immune response to the composition.

A “subject” refers to a donor, recipient or host of the composition ofthe present invention. In some embodiments, the donor and the recipientare the same. In some embodiments the subject is a human subject.

A “proteoglycan” refers to a special class of proteins that are heavilyglycosylated. A proteoglycan is made up of a core protein with numerouscovalently attached high sulphated glycosaminoglycan chain(s).Non-limiting example of extracellular matrix proteoglycans includeaggrecan and certain collagens, such as collagen IX.

A “glycosaminoglycan” or “GAG” as used herein refers to a longunbranched polysaccharide molecules found on the cell surface or withinthe extracellular matrix. Non-limiting examples of glycosaminoglycaninclude heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate,heparan sulfate, keratan sulfate, hyaluronic acid, hexuronylhexosaminoglycan sulfate, and inositol hexasulfate.

The term “non-autologous” refers to tissue or cells which originate froma donor other than the recipient. Non-autologous can refer to, forexample, allogeneic or xenogeneic. The term “autologous” as in anautologous composition, refers to a composition in which the donor andrecipient is the same individual. Likewise, “allogeneic” refers to adonor and a recipient of the same species; “syngeneic” refers to a donorand recipient with identical genetic make-up (e.g., identical twins orautogenic) and “xenogeneic” refers to donor and recipient of differentspecies.

The term “variant” (or “analog”) refers to any molecule differing fromthe naturally occurring molecule.

The term “variant polynucleotide” (or “analog”) refers to anypolynucleotide differing from the naturally occurring polynucleotide.For example, “variant A2M polynucleotide” refers to any A2Mpolynucleotide differing from naturally occurring A2M polynucleotides. Avariant A2M polynucleotide includes a polynucleotide sequence differentfrom the wild-type A2M polynucleotide sequence (SEQ ID NO: 1). Variantpolynucleotides can be characterized by nucleic acid insertions,deletions, and substitutions, created using, for example, recombinantDNA techniques. A variant A2M polynucleotide preferably includes amutation, insertion, deletion, or a combination thereof, in the baitregion of a wild-type A2M polynucleotide sequence. As used herein, whenreferring to polypeptides, the “bait region” includes the region of anA2M polynucleotide that encodes the region of the A2M polypeptide thatbinds to proteases, for example, regions that contain proteaserecognition sites. A variant A2M polynucleotide includes an “A2Macceptor sequence” (SEQ ID NO: 2) which includes a polynucleotidesequence of A2M with point mutations that can aid in creating variantA2M polynucleotides by recombinant DNA techniques, for example, bycreating restriction enzyme cloning sites to aid in inserting variouspolynucleotide sequences encoding the variant bait regions. Bait regionsinclude SEQ ID NOs: 5-66 and sequences substantially similar to SEQ IDNOs: 5-66.

The term “variant polypeptide” refers to any polypeptide differing fromthe naturally occurring polypeptide. For example, “variant A2Mpolypeptide” refers to any A2M polypeptide differing from naturallyoccurring A2M polypeptides. Variant polypeptides can be characterized byamino acid insertions, deletions, and substitutions, created using, forexample, recombinant DNA techniques. A variant A2M polypeptide includesa polypeptide sequence different from the wild-type A2M polypeptidesequence. A variant A2M polypeptide preferably includes a mutation,insertion, deletion, or a combination thereof, in the bait region of awild-type A2M protein. When referring to polypeptides, the “bait region”includes the region of an A2M polypeptide that binds to proteases, forexample, a stretch of amino acids that contains one or more proteaserecognition sites. A variant A2M polypeptide includes a polypeptide (SEQID NO: 3) encoded by an A2M acceptor sequence (SEQ ID NO: 2). A “variantA2M polypeptide” can have at least one amino acid sequence alteration inthe bait region as compared to the amino acid sequence of thecorresponding wild-type polypeptide. An amino acid sequence alterationcan be, for example, a substitution, a deletion, or an insertion of oneor more amino acids. A variant A2M polypeptide can have any combinationof amino acid substitutions, deletions or insertions.

Guidance in determining which amino acid residues may be replaced, addedor deleted without abolishing activities of interest, may be found bycomparing the sequence of the particular polypeptide with that ofhomologous peptides and minimizing the number of amino acid sequencechanges made in regions of high homology (conserved regions) or byreplacing amino acids with consensus sequence. Alternatively,recombinant variants encoding these same or similar polypeptides may besynthesized or selected by making use of the “redundancy” in the geneticcode. Various codon substitutions, such as the silent changes whichproduce various restriction sites, may be introduced to optimize cloninginto a plasmid or viral vector or expression in a particular prokaryoticor eukaryotic system. Mutations in the polynucleotide sequence may bereflected in the polypeptide or domains of other peptides added to thepolypeptide to modify the properties of any part of the polypeptide, tochange characteristics such as inhibition of proteases, ligand-bindingaffinities, interchain affinities, or degradation/turnover rate. Variantnucleotides can also be used to generate polypeptides that are bettersuited for expression, scale up and the like in the host cells chosenfor expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

An amino acid “substitution” includes replacing one amino acid withanother amino acid having similar structural and/or chemical properties,for example, conservative amino acid replacements. “Conservative” aminoacid substitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, the amphipathicnature of the residues involved, or a combination thereof. Nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. Polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine Positively charged (basic) amino acids includearginine, lysine, and histidine. Negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. “Insertions” or “deletions” arepreferably in the range of about 1 to 50 amino acids, more preferably 1to 30 amino acids. The variation allowed can be experimentallydetermined by inserting, deleting, or substituting amino acids in apolypeptide using recombinant DNA techniques and assaying the resultingrecombinant variants for activity, for example, protease inhibitionactivity.

The terms “purified” or “substantially purified” as used herein denotesthat the indicated nucleic acid or polypeptide is present in thesubstantial absence of other biological macromolecules, for example,polynucleotides, proteins, and the like. The polynucleotide orpolypeptide can be purified such that it constitutes at least 95% byweight, for example, at least 99% by weight, of the indicated biologicalmacromolecules present. Water, buffers, and other small molecules with amolecular weight of less than 1000 Daltons, can be present in anyamount. The term “isolated” as used herein refers to a polynucleotide orpolypeptide separated from at least one other component present with thepolynucleotide or polypeptide in its natural source. In someembodiments, the polynucleotide or polypeptide can be found in thepresence of only a solvent, buffer, ion, or other components normallypresent in a solution of the same. The terms “isolated” and “purified”do not encompass polynucleotides or polypeptides present in theirnatural source.

As used herein, “recombinant polypeptides” include polypeptides orproteins derived from recombinant expression systems, for example,microbial, insect, or mammalian expression systems. Polypeptides orproteins expressed in most bacterial cultures will be free ofglycosylation modifications; polypeptides or proteins expressed in yeastcan have a glycosylation pattern in general different from thoseexpressed in mammalian cells.

The term “expression vector” refers to a plasmid or phage or virus orvector, for expressing a polypeptide from a DNA or RNA sequence. Anexpression vector can include a transcriptional unit comprising anassembly of a genetic element or elements having a regulatory role ingene expression, for example, promoters or enhancers, a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems can include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an amino terminal methionine residue. This residue may ormay not be subsequently cleaved from the expressed recombinant proteinto provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems can be used to express heterologouspolypeptides or proteins upon induction of the regulatory elementslinked to the DNA segment or synthetic gene to be expressed. This termincludes host cells which have stably integrated a recombinant geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers. Recombinant expression systems can beused to express polypeptides or proteins endogenous to the cell uponinduction of the regulatory elements linked to the endogenous DNAsegment or gene to be expressed. The cells can be prokaryotic oreukaryotic.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly,for example soluble proteins, or partially, for example receptors, fromthe cell in which they are expressed. “Secreted” proteins also includeproteins transported across the membrane of the endoplasmic reticulum.“Secreted” proteins also include proteins containing non-typical signalsequences.

An expression vector may be designed to contain a “signal sequence”which will direct the polypeptide through the membrane of a cell. Asignal sequence can be naturally present on the polypeptides describedherein or provided from heterologous protein sources.

As used herein, “substantially equivalent” or “substantially similar”can refer both to nucleotide and amino acid sequences, for example avariant sequence, that varies from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which does notresult in an adverse functional dissimilarity between the reference andsubject sequences. Typically, such a substantially equivalent sequencevaries from one of those listed herein by no more than about 35%. Forexample, the number of individual residue substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.35 or less.A substantially equivalent sequence includes sequences with 65% sequenceidentity to the reference sequence. A substantially equivalent sequenceof the invention can vary from a reference sequence by no more than 30%(70% sequence identity), no more than 25% (75% sequence identity), nomore than 20% (80% sequence identity), no more than 10% (90% sequenceidentity), or no more that 5% (95% sequence identity). Substantiallyequivalent amino acid sequences according to the invention preferablyhave at least 80% sequence identity with a reference amino acidsequence, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity. Substantially equivalentpolynucleotide sequences of the invention can have lower percentsequence identities, taking into account, for example, the redundancy ordegeneracy of the genetic code. Preferably, the polynucleotide sequencehas at least about 65%, at least about 75%, at least about 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. Sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. Identity between sequences can be determinedby methods known in the art, such as by alignment of the sequences orvarying hybridization conditions.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviatechronic wounds such as pressure ulcers, venous ulcers, stasis ulcers,venous stasis ulcers, diabetic foot ulcers, arterial insufficiencyulcers or any combination thereof in a subject in need thereof.

By “degenerate variant” can be intended nucleotide fragments whichdiffer from a nucleic acid fragment of the present invention (e.g., anORF) by nucleotide sequence but, due to the degeneracy of the geneticcode, encode an identical polypeptide sequence.

The terms polypeptide, peptide, and protein can be used interchangeablyand can refer to a polymer of amino acid residues or a variant thereof.Amino acid polymers can have one or more amino acid residues and can bean artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers, thosecontaining modified residues, and non-naturally occurring amino acidpolymers. A variant polypeptide can have at least one amino acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide. An amino acid sequence alterationcan be, for example, a substitution, a deletion, or an insertion of oneor more amino acids. A variant polypeptide can have any combination ofamino acid substitutions, deletions or insertions. An amino acidsequence alteration can be formed by altering the nucleotide sequencefrom which it is derived, such as a mutation, for example, a frameshiftmutation, nonsense mutation, missense mutation, neutral mutation, orsilent mutation. For example, sequence differences, when compared to awild-type nucleotide sequence, can include the insertion or deletion ofa single nucleotide, or of more than one nucleotide, resulting in aframe shift; the change of at least one nucleotide, resulting in achange in the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of a reading frame; duplication ofall or a part of a sequence; transposition; or a rearrangement of anucleotide sequence. Such sequence changes can alter the polypeptideencoded by the nucleic acid, for example, if the change in the nucleicacid sequence causes a frame shift, the frame shift can result in achange in the encoded amino acids, and/or can result in the generationof a premature stop codon, causing generation of a truncatedpolypeptide.

The term “fragment” can refer to any subset of the polypeptide that canbe a shorter polypeptide of the full length protein. Fragments of A2Mcan include 20, 30, 40, 50 or more amino acids from A2M that can bedetected with anti-A2M antibodies. Other fragments of A2M includevarious domains of A2M and combinations thereof.

“Platelet-rich plasma” (“PRP”) can refer to blood plasma that has beenenriched with platelets.

“Platelet-poor plasma” (“PPP”) can refer to blood plasma that has beendepleted of platelets.

Wounds and Wound Classification

Chronic wounds, slow healing wounds, and incomplete healing wounds oftenresult in infection and can lead to amputation or death. It has beendiscovered that use of certain compounds, including those described orreferenced herein, may block, inhibit, or alter cell communications,which may promote closure and healing in chronic, slow healing, andincomplete healing wounds.

By “wound” is meant an injury to any tissue, including, for example,acute, delayed, slow, or difficult to heal wounds, and chronic wounds.Examples of wounds may include both open and closed wounds. Woundsinclude, for example, burns, incisions, excisions, lacerations,abrasions, puncture or penetrating wounds, surgical wounds, contusions,hematomas, crushing injuries, and ulcers. Also included are wounds thatdo not heal at expected rates.

By a “slow healing wound” is meant an injury to any tissue that does notheal in an expected or typical time frame, including delayed, slow, ordifficult to heal wounds (including delayed or incompletely healingwounds), and chronic wounds. Examples of wounds that do not heal at theexpected rate include diabetic ulcers, diabetic foot ulcers, vasculiticulcers, arterial ulcers, venous ulcers, venous stasis ulcers, pressureulcers, decubitus ulcers, infectious ulcers, trauma-induced ulcers, burnulcers, ulcerations associated with pyoderma gangrenosum, and mixedulcers.

As described herein, a slow healing wound may include, for example, awound that is characterized at least in part by one or more of 1) aprolonged inflammatory phase, 2) a slow forming extracellular matrix,and 3) a stalled or decreased rate of epithelialization.

In the art, the term “chronic wound” refers generally to a wound thathas not healed within about three months, but can be wounds that havenot healed within about one or two months. Chronic skin wounds include,for example, pressure ulcers, diabetic ulcers, venous ulcers, arterialulcers, inflammatory ulcers, and mixed ulcers. The chronic wound may bean arterial ulcer that can include ulcerations resulting from completeor partial arterial blockage. The chronic wound may be a venous stasisulcer, which can include ulcerations resulting from a malfunction of thevenous valve and the associated vascular disease. The chronic wound maybe a trauma-induced ulcer.

As used herein, chronic wound can also include, for example, a woundthat is characterized at least in part by 1) a chronic self-perpetuatingstate of wound inflammation, 2) a deficient and defective woundextracellular matrix (ECM), 3) poorly responding (senescent) wound cells(e.g., fibroblasts), limited ECM production, and 4) failure ofre-epithelialization due in part to lack of the necessary ECMorchestration and lack of scaffold for migration.

Chronic wounds can also be characterized, for example, by 1) prolongedinflammation and proteolytic activity, leading to ulcerative lesions,including, for example, diabetic, pressure (decubitus), venous, andarterial ulcers, 2) prolonged fibrosis in the wound leading to scarring,3) progressive deposition of matrix in the affected area, 4) longerrepair times, 5) less wound contraction, 6) slower re-epithelialization,and 7) increased thickness of granulation tissue.

Exemplary chronic wounds also include “pressure ulcers.” Exemplarypressure ulcers may include all four stages of wound classificationsbased on AHCPR (Agency for Health Care Policy and Research, U.S.Department of Health and Human Services) guidelines, including forexample, Stage 1. A Stage 1 pressure ulcer is an observable pressurerelated alteration of intact skin whose indicators as compared to theadjacent or opposite area on the body may include changes in one or moreof the following: skin temperature (warmth or coolness), tissueconsistency (firm or boggy feel), and/or sensation (pain, itching). Theulcer appears as a defined area of persistent redness in lightlypigmented skin, whereas in darker skin tones, the ulcer may appear withpersistent red, blue, or purple hues. Stage 1 ulcerations may includenon-blanchable erythema of intact skin and the heralding lesion of skinulceration. In individuals with darker skin, discoloration of the skin,warmth, edema, induration, or hardness may also be indicators of stage 1ulcerations. Stage 2 ulcerations may be characterized by partialthickness skin loss involving epidermis, dermis, or both. The ulcer issuperficial and presents clinically as an abrasion, blister, or shallowcrater. Stage 3 ulcerations may be characterized by full thickness skinloss involving damage to or necrosis of subcutaneous tissue that mayextend down to, but not through, underlying fascia. The ulcer presentsclinically as a deep crater with or without undermining of adjacenttissue. Stage 4 ulcerations may be characterized by full thickness skinloss with extensive destruction, tissue necrosis, or damage to muscle,bone, or supporting structures (e.g., tendon, joint capsule, etc.).

Exemplary chronic wounds also include “decubitus ulcers.” Exemplarydecubitus ulcer may arise as a result of prolonged and unrelievedpressure over a bony prominence that leads to ischemia. The wound tendsto occur in patients who are unable to reposition themselves to off-loadweight, such as paralyzed, unconscious, or severely debilitated persons.As defined by the U.S. Department of Health and Human Services, themajor preventive measures include identification of high-risk patients;frequent assessment; and prophylactic measures such as scheduledrepositioning, appropriate pressure-relief bedding, moisture barriers,and adequate nutritional status. Treatment options may include, forexample, pressure relief, surgical and enzymatic debridement, moistwound care, and bacterial load control. Certain embodiments of theinvention involve treating a chronic wound characterized by a decubitusulcer or ulceration that results from prolonged, unrelieved pressureover a bony prominence that leads to ischemia.

Exemplary chronic wounds also include “arterial ulcers.” Arterial ulcersinclude those characterized by complete or partial arterial blockage,which may lead to tissue necrosis and/or ulceration. Signs of arterialulcer can include, for example, pulselessness of the extremity; painfululceration; small, punctuate ulcers that are usually well circumscribed;cool or cold skin; delayed capillary return time (briefly push on theend of the toe and release, normal color should return to the toe inabout 3 seconds or less); atrophic-appearing skin (for example, shiny,thin, dry); and loss of digital and pedal hair.

Exemplary chronic wounds also include “venous ulcers.” Exemplary venousulcers include the most common type of ulcer affecting the lowerextremities and may be characterized by malfunction of the venous valve.The normal vein has valves that prevent the backflow of blood. Whenthese valves become incompetent, the backflow of venous blood causesvenous congestion. Hemoglobin from the red blood cells escapes and leaksinto the extravascular space, causing the brownish discolorationcommonly noted. It has been shown that the transcutaneous oxygenpressure of the skin surrounding a venous ulcer is decreased, indicatingthat there are forces obstructing the normal vascularity of the area.Lymphatic drainage and flow also plays a role in these ulcers. A venousulcer can appear near the medial malleolus and usually occurs incombination with an edematous and indurated lower extremity; it may beshallow, not too painful, and may present with a weeping discharge fromthe affected site.

Exemplary chronic wounds also include “venous stasis ulcers.” Exemplaryvenous stasis ulcers are characterized by chronic passive venouscongestion of the lower extremities that results in local hypoxia. Onepossible mechanism of pathogenesis of these wounds includes theimpediment of oxygen diffusion into the tissue across thick perivascularfibrin cuffs. Another mechanism is that macromolecules leaking into theperivascular tissue trap growth factors needed for the maintenance ofskin integrity. Additionally, the flow of large white blood cells slowsdue to venous congestion, occluding capillaries, becoming activated, anddamaging the vascular endothelium to predispose to ulcer formation.

Exemplary chronic wounds further include “diabetic foot ulcers.”Diabetic patients with exemplary diabetic foot ulcer are prone to footulcerations due to both neurologic and vascular complications.Peripheral neuropathy can cause altered or complete loss of sensation inthe foot and/or leg. Diabetic patients with advanced neuropathy lose allability for sharp-dull discrimination. Any cuts or trauma to the footmay go completely unnoticed for days or weeks in a patient withneuropathy. A patient with advanced neuropathy can lose the ability tosense a sustained pressure insult and, as a result, tissue ischemia andnecrosis may occur leading to, for example, plantar ulcerations.Additionally, microfractures in the bones of the foot, if unnoticed anduntreated, may result in disfigurement, chronic swelling, and additionalbony prominences. Microvascular disease is one of the significantcomplications for diabetics that may also lead to ulcerations.

Exemplary chronic wounds can include “traumatic ulcers.” Formation ofexemplary traumatic ulcers may occur as a result of traumatic injuriesto the body. These injuries include, for example, compromises to thearterial, venous, or lymphatic systems; changes to the bony architectureof the skeleton; loss of tissue layers—epidermis, dermis, subcutaneoussoft tissue, muscle or bone; damage to body parts or organs and loss ofbody parts or organs.

Exemplary chronic wounds can include “burn ulcers” including, forexample, ulceration that occur as a result of a burn injury, including afirst degree burn (i.e., superficial, reddened area of skin); a seconddegree burn (a blistered injury site which may heal spontaneously afterthe blister fluid has been removed); a third degree burn (burn throughthe entire skin and usually require surgical intervention for woundhealing); scalding (may occur from scalding hot water, grease orradiator fluid); a thermal burn (may occur from flames, usually deepburns); a chemical burn (may come from acid and alkali, usually deepburns); an electrical burn (either low voltage around a house or highvoltage at work); an explosion flash (usually superficial injuries); andcontact burns (usually deep and may occur from muffler tail pipes, hotirons, and stoves).

Compositions for Autologous Treatment of Wounds

Provided herein are autologous compositions. The autologous compositionscan be prepared from a biological sample from a subject and administeredback to the same subject.

Also provided herein are therapeutic autologous compositions and methodsfor their preparation and use. Also provided herein are methods,systems, kits and compositions for the detection, diagnosis, andtreatment of inflammation, ECM degradation, and chronic wounds such aspressure ulcers, venous ulcers, stasis ulcers, venous stasis ulcers,diabetic foot ulcers, arterial insufficiency ulcers or any combinationthereof.

An autologous composition can comprise alpha-2-macroglobulin (A2M) andcan be used to treat a subject with a condition. The A2M can be from abiological sample, such as from a human subject; or can be any fragmentthereof. In preferred embodiments the autologous compositions of thepresent invention are substantially non-immunogenic, namely do notelicit an immune response.

An autologous composition can comprise an elevated concentration of A2Mcompared to the concentration of A2M found in a biological sample, suchas the blood of normal subjects, or the blood from a subject in need ofautologous treatment with the autologous composition. The concentrationof A2M in an autologous composition can be at least about 1.1 timeshigher than the concentration of A2M found in a biological sample. Forexample, the concentration of A2M can be at least about 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higherthan the concentration of A2M found in a biological sample. For example,the concentration of A2M can be at least about 2 times higher than theconcentration of A2M found in the biological sample.

In some embodiments, an autologous composition can further comprise areduced concentration of components other than A2M compared to thenormal concentration of the other components, such as the concentrationin a sample from which the autologous compositions were prepared or anendogenous concentration of the other components in a biological sample.An autologous composition can comprise a reduced concentration of othercomponents isolated from a biological sample compared to the normalconcentration of the other components in the biological sample. Theconcentration of other components can be at least about 10% less thanthe concentration of the other components normally found in a biologicalsample. For example, the concentration of other components can be atleast about 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, or 1000% less than the concentrationof the other components normally found in a biological sample. Forexample, the concentration of other components can be at least about 20%less than the concentration of the other components normally found in abiological sample. The concentration of other components can be at leastabout 0.1 times less than the concentration of the other componentsnormally found in a biological sample. For example, the concentration ofother components can be at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times less than the concentration of theother components normally found in a biological sample. For example, theconcentration of other components can be at least about 2 times lessthan the concentration of the other components normally found in abiological sample.

In some embodiments, an autologous composition can comprise a reducedconcentration of one or more proteins with a molecular weight lower than50, 100, and/or 500 kDa. The concentration of one or more proteins witha molecular weight lower than 500 kDa can be at least about 1.1 timeslower than the concentration of the one or more proteins with molecularweight lower than 50, 100, and/or 500 kDa found in a normal biologicalsample, such as blood from a subject. The concentration of one or moreproteins with a molecular weight lower than 50, 100, and/or 500 kDafound in a normal biological sample can be the concentration of theendogenous level of the one or more proteins with a molecular weightlower than 50, 100, and/or 500 kDa in a biological sample, such as anormal or control biological sample. For example, the concentration ofone or more proteins with molecular weight lower than 100 kDa can be atleast about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 timeslower than the concentration of the one or more proteins with molecularweight lower than 50, 100, and/or 500 kDa found in a normal biologicalsample or the endogenous concentration in a normal biological sample, orthe blood from a subject in need of autologous treatment with theautologous composition. For example, the concentration of one or moreproteins with molecular weight lower than 50, 100, and/or 500 kDa can beat least about 1.5 times lower than the concentration of the one or moreproteins with molecular weight lower than 50, 100, and/or 500 kDa foundin a normal biological sample or the endogenous concentration in anormal biological sample.

In some embodiments, an autologous composition can comprise an elevatedconcentration of one or more proteins with a molecular weight higherthan 50, 100, and/or 500 kDa than normally found in a biological sample.The concentration of one or more proteins with a molecular weight higherthan 50, 100, and/or 500 kDa can be at least about 1.1 times higher thanthe concentration of the one or more proteins with molecular weighthigher than 50, 100, and/or 500 kDa found in a normal biological sample.The concentration of one or more proteins with a molecular weight higherthan 50, 100, and/or 500 kDa found in a normal biological sample can bethe concentration of the endogenous level of the one or more proteinswith a molecular weight higher than 50, 100, and/or 500 kDa in thebiological sample. For example, the concentration of one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa canbe at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times higher than the concentration of the one or more proteins withmolecular weight higher than 50, 100, and/or 500 kDa found in a normalbiological sample or the endogenous concentration in a normal biologicalsample. For example, the concentration of one or more proteins withmolecular weight higher than 50, 100, and/or 500 kDa can be at leastabout 1.5 times higher than the concentration of the one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa foundin a normal biological.

In some embodiments, proteins with a molecular weight from about 300-500kDa, 100-500 kDa, and/or 50-500 kDa, such as fibronectin, fibrinogen,and fibrin monomers or polymers may be partially concentrated using themethods described herein. In some embodiments, an autologous compositioncan comprise an elevated concentration of one or more proteins with amolecular weight from about 300-500 kDa, 100-500 kDa, and/or 50-500 kDa.In some embodiments, the concentration of one or more proteins with amolecular weight higher than from about 300-500 kDa, 100-500 kDa, and/or50-500 kDa can be at least about 1.1 times higher than the concentrationof the one or more proteins with molecular weight from about 300-500kDa, 100-500 kDa, and/or 50-500 kDa found in a normal biological sample,such as blood from a subject. The concentration of one or more proteinswith a molecular weight from about 300-500 kDa, 100-500 kDa, and/or50-500 kDa found in a normal biological sample can be the concentrationof the endogenous level of the one or more proteins with a molecularweight from about 300-500 kDa, 100-500 kDa, and/or 50-500 kDa in abiological sample, such as a normal or control biological sample. Forexample, the concentration of one or more proteins with molecular weightfrom about 300-500 kDa, 100-500 kDa, and/or 50-500 kDa can be at leastabout 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higherthan the concentration of the one or more proteins with molecular weightfrom about 300-500 kDa, 100-500 kDa, and/or 50-500 kDa found in a normalbiological sample or the endogenous concentration in a normal biologicalsample.

An autologous composition can comprise an elevated concentration of A2Mcompared to the concentration of A2M found in a biological sample and areduced concentration of components other than A2M compared to thenormal concentration of the other components found in a biologicalsample. The concentration of A2M in an autologous composition can be atleast about 1.1 times higher than the concentration of A2M found in abiological sample and the concentration of other components other thanA2M can be at least about 0.1 times less than the concentration of theother components normally found in a biological sample. For example, theconcentration of A2M can be at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of A2Mfound in a biological sample and the concentration of components otherthan A2M can be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, or 1000 times less than the concentration of the othercomponents normally found in a biological sample. For example, theconcentration of the concentration of A2M can be at least about 2 timeshigher than the concentration of A2M found in a biological sample andthe concentration of components other than A2M can be at least about 2times less than the concentration of the other components normally foundin a biological sample. As another example, the concentration of A2M canbe at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times higher than the concentration of A2M found in a biological sampleand the concentration of other components can be at least about 11%,12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%,700%, 800%, 900%, or 1000% less than the concentration of the othercomponents normally found in a biological sample. For example, theconcentration of A2M can be at least about 2 times higher than theconcentration of A2M found in a biological sample and the concentrationof other components can be at least about 20% less than theconcentration of the other components normally found in a biologicalsample.

In some embodiments, an autologous composition can comprise an elevatedconcentration of A2M compared to the concentration of A2M found in abiological sample and an elevated concentration of one or more proteinswith molecular weight higher than 50, 100, and/or 500 kDa found in abiological sample. The concentration of A2M in an autologous compositioncan be at least about 1.1 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa canbe at least about 1.1 times higher than the concentration of the one ormore proteins with molecular weight higher than 50, 100, and/or 500 kDafound in a biological sample. For example, the concentration of theconcentration of A2M can be at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa canbe at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times higher than the concentration of the one or more proteins withmolecular weight higher than about 50, 100, and/or 500 kDa found in abiological sample. For example, the concentration of A2M can be at leastabout 2 times higher than the concentration of A2M found in a biologicalsample and the concentration of one or more proteins with molecularweight higher than 50, 100, and/or 500 kDa can be at least about 2 timeshigher than the concentration of the one or more proteins with molecularweight higher than about 100 kDa found in a biological sample.

The concentration of A2M and other proteins with a molecular weighthigher than 50, 100, and/or 500 kDa can be present at a concentration ofat least about 1.1 times higher than their concentration in a biologicalsample after retention by one or more filters using the methods orsystems described herein. For example, the concentration of A2M andother proteins with a molecular weight higher than 50, 100, and/or 500kDa can be present at a concentration of at least about 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein. For example, theconcentration of A2M and other proteins with a molecular weight higherthan 50, 100, and/or 500 kDa can be present at a concentration of atleast about 1.5 times higher than their concentration in a biologicalsample after retention by one or more filters using the methods orsystems described herein.

The concentration of proteins with molecular weight less than about 50,100, and/or 500 kDa can be less than about 10% of the concentrations ofthose proteins in a biological sample when retained by the one or morefilters using the methods or systems described herein. For example, theconcentration of proteins with molecular weight less than about 50, 100,and/or 500 kDa can be less than about 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%less than their concentration in a biological sample when retained bythe one or more filters using the methods or systems described herein.For example, the concentration of proteins with molecular weight lessthan about 50, 100, and/or 500 kDa can be less than about 20% less thantheir concentration in a biological sample when retained by the one ormore filters using the methods or systems described herein.

In some embodiments, the concentration of proteins with molecular weightless than about 50, 100, and/or 500 kDa can be less than about 10% ofthe concentrations of those proteins in a biological sample whenretained by the one or more filters. For example, the concentration ofproteins with molecular weight less than about 50, 100, and/or 500 kDacan be less than about 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% less than theirconcentration in a biological sample when retained by the one or morefilters. For example, the concentration of proteins with molecularweight less than about 50, 100, and/or 500 kDa can be less than about20% less than their concentration in a biological sample when retainedby the one or more filters.

The concentration of A2M and other proteins with a molecular weighthigher than 50, 100, and/or 500 kDa can be present at a concentration ofat least about 1.1 times higher than their concentration in a biologicalsample after retention by one or more filters using the methods orsystems described herein and the concentration of proteins withmolecular weight less than about 50, 100, and/or 500 kDa can be lessthan about 10% of the concentrations of those proteins in a biologicalsample when retained by the one or more filters using the methods orsystems described herein. For example, the concentration of A2M andother proteins with a molecular weight higher than 50, 100, and/or 500kDa can be present at a concentration of at least about 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein, and theconcentration of proteins with molecular weight less than about 50, 100,and/or 500 kDa can be less than about 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%less than their concentration in a biological sample when retained bythe one or more filters using the methods or systems described herein.For example, the concentration of A2M and other proteins with amolecular weight higher than 50, 100, and/or 500 kDa can be present at aconcentration of at least about 1.5 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein, and theconcentration of proteins with molecular weight less than about 50, 100,and/or 500 kDa can be less than about 10% less than their concentrationin a biological sample when retained by the one or more filters.

Proteins with a molecular weight higher than about 100 kDa can be, forexample, proteins with a molecular weight at least about 110 kDa, 120kDa, 130 kDa, 140 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa,1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa,1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa,1900 kDa, 1950 kDa, 2000 kDa, or more.

Proteins with a molecular weight less than about 100 kDa can be, forexample, proteins with a molecular weight of less than about 95 kDa, 90kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa, 55 kDa, 50 kDa, 45kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa, 5 kDa, orless.

Proteins with a molecular weight higher than about 500 kDa can be, forexample, proteins with a molecular weight higher than about 550 kDa, 600kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, orhigher. Proteins with a molecular weight less than about 500 kDa can be,for example, proteins with a molecular weight less than about 450 kDa,400 kDa, 350 kDa, 300 kDa, 250 kDa, 200 kDa, 150 kDa, 100 kDa, 50 kDa,45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa, 5 kDa,or less.

Proteins with a molecular weight higher than 100 kDa can include, butare not limited to, immunoglobulin G, immunoglobulin M, fibronectin,fibrinogen and other proteins. Proteins with a molecular weight lessthan about 100 kDa can comprise cytokines, chemokines, proteases,pro-proteases, enzymes, pro-enzymes, immune-modulators and otherproteins known in the art with a molecular weight of less than 100 kDa.

Proteins with a molecular weight higher than 500 kDa include, but arenot limited to, IgM and Complement Component C4 binding proteins.Proteins with a molecular weight less than about 500 kDa can comprisecytokines, chemokines, proteases, pro-proteases, enzymes, pro-enzymes,immune-modulators and other proteins with a molecular weight of lessthan 500 kDa.

The concentration of A2M found in a biological sample, such as a bloodsample from a normal subject, can be between about 0.1 mg/mL to about 6mg/mL. For example, the concentration of A2M found in a blood samplefrom a normal subject or a normal biological sample can be between about0.1 mg/mL to 5.5 mg/mL, 0.1 mg/mL to 5 mg/mL, 0.1 mg/mL to 4.5 mg/mL,0.1 mg/mL to 4 mg/mL, 0.1 mg/mL to 3.5 mg/mL, 0.1 mg/mL to 3 mg/mL, 0.1mg/mL to 2.5 mg/mL, 0.1 mg/mL to 2 mg/mL, 0.1 mg/mL to 1.5 mg/mL, 0.1mg/mL to 1 mg/mL, 0.1 mg/mL to 0.75 mg/mL, 0.1 mg/mL to 0.5 mg/mL, 0.1mg/mL to 0.25 mg/mL, 1 mg/mL to 6 mg/mL, 1 mg/mL to 5.5 mg/mL, 1 mg/mLto 5 mg/mL, 1 mg/mL to 4.5 mg/mL, 1 mg/mL to 4 mg/mL, 1 mg/mL to 3.5mg/mL, 1 mg/mL to 3 mg/mL, 1 mg/mL to 2.5 mg/mL, 1 mg/mL to 2 mg/mL, 1mg/mL to 1.5 mg/mL, 2 mg/mL to 6 mg/mL, 2 mg/mL to 5.5 mg/mL, 2 mg/mL to5 mg/mL, 2 mg/mL to 4.5 mg/mL, 2 mg/mL to 4 mg/mL, 2 mg/mL to 3.5 mg/mL,2 mg/mL to 3 mg/mL, 2 mg/mL to 2.5 mg/mL, 3 mg/mL to 6 mg/mL, 3 mg/mL to5.5 mg/mL, 3 mg/mL to 5 mg/mL, 3 mg/mL to 4.5 mg/mL, 3 mg/mL to 4 mg/mL,3 mg/mL to 3.5 mg/mL, 4 mg/mL to 6 mg/mL, 4 mg/mL to 5.5 mg/mL, 4 mg/mLto 5 mg/mL, 4 mg/mL to 4.5 mg/mL, 5 mg/mL to 6 mg/mL, or 5 mg/mL to 5.5mg/mL.

In some embodiments, an autologous composition with an elevatedconcentration of A2M can be characterized by a reduction in theconcentration of or a change in the ratios of cytokines, chemokines,other immunomodulatory mediators, for example, cytokines, chemokines,other immunomodulatory mediators with a molecular weight less than about50, 100, and/or 500 kDa. In some embodiments, a retentate with anelevated concentration of A2M can be characterized by a reduction in theconcentration of or a change in the ratios of cytokines, chemokines,other immunomodulatory mediators, for example, cytokines, chemokines,other immunomodulatory mediators with a molecular weight less than about50, 100, and/or 500 kDa. Other immunomodulatory mediators can includepeptides, proteins, DNA, RNA, carbohydrates, other small molecules,proteases, and other degradative proteins.

Cytokines, chemokines and other molecules in a composition with areduction in concentration from a sample can be involved ininflammation. Cytokines can be small cell-signaling protein moleculesthat are secreted by one or more cells and are a category of signalingmolecules that can be used in intercellular communication. Cytokines canbe classified as proteins, peptides, or glycoproteins, chemokines,interleukins, tumor necrosis factors (TNFs), monocyte chemoattractantproteins (MCPs), IL-1-like cytokines, gamma chain cytokines, beta chaincytokines, IL-6-like cytokines, IL-10-like cytokines, interferons, tumornecrosis factors, TGF-beta, macrophage inflammatory proteins (MIPs),tumor growth factors (TGFs), and matrix metalloproteases (MMPs). Forexample, cytokines can be interleukins, such as IL-1-like, IL-1α(hematopoietin-1), IL-1β (catabolin), IL-1RA (IL-1 receptor antagonist),IL-18 (interferon-γ inducing factor), Common g chain (CD132), IL-2 (Tcell growth factor), IL-4 (BSF-1), IL-7, IL-9 (T cell growth factorP40), IL-13 (P600), IL-15, Common b chain (CD131), IL-3 (multipotentialCSF, MCGF), IL-5 (BCDF-1), GM-CSF (CSF-2), IL-6-like, IL-6 (IFN-β2,BSF-2), IL-11 (AGIF), G-CSF (CSF-3), IL-12 (NK cell stimulatory factor),LIF (leukemia inhibitory factor), OSM (oncostatin M), IL-10-like, IL-10(CSIF), IL-20, IL-14 (HMW-BCGF), IL-16 (LCF), and IL-17 (CTLA-8);interferons, such as IFN-α, IFN-β, and IFN-γ, tumor necrosis factors(TNFs), such as CD154 (CD40L, TRAP), LT-β, TNF-α (cachectin), TNF-β(LT-α), 4-1BBL, APRIL (TALL-2), CD70 (CD27L), CD153 (CD30L), CD178(FasL), GITRL, LIGHT, OX40L, TALL-1, TRAIL (Apo2L), TWEAK (Apo3L), andTRANCE (OPGL); tumor growth factors, such as TGF-β1, (TGF-β), TGF-β2,and TGF-β3; and hematopoietins, such as Epo (erythropoietin), Tpo(MGDF), Flt-3L, SCF (stem cell factor, c-kit ligand), M-CSF (CSF-1), andMSP (Macrophage stimulating factor). Other cytokines can include MST-1,CD40LG (TNFSF5), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL36RN,IL36A, IL37, IL36B, IL36G, IL22, IL5, IL8, IL9, LTA, LTB, MIF, AIMP1,SPP1, and TNF. Exemplary, cytokine receptors can be IFNA2, IL10RA,IL10RB, IL13, IL13RA1, IL5RA, IL9, and IL9R.

Chemokines can be a family of small cytokines, or proteins secreted bycells. Some chemokines can be pro-inflammatory and can be induced duringan immune response to recruit cells of an immune system to a site ofinfection, while others can be homeostatic and can be involved incontrolling the migration of cells during normal processes of tissuemaintenance or development. For example, chemokines can be XCL1(lymphoactin a, SCM-1a, ATAC), XCL2 (lymphoactin b, SCM-1b, ATAC,) CCL1(I-309), CCL2 (MCP-1, MCAF), CCL3 (MIP-1α, LD78α), CCL4 (MIP-1β, LAG-1,ACT-2), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (eotaxin),CCL13 (MCP-4), CCL14 (HCC-1), CCL15 (HCC-2, Lkn-1, MIP-1d, MIP-5), CCL16(HCC-4, LEC, LMC, LCC-1), CCL17 (TARC), CCL18 (DC-CK1, PARC, AMAC-a,MIP-4), CCL19 (MIP-3β, ELC, exodus-3), CCL20 (MIP-3α, LARC, exodus-1),CCL21 (6Ckine, SLC, exodus-2), CCL22 (MDC, STCP-1), CCL23 (MPIF-1,MIP-3, CKb-8), CCL24 (MPIF-2, eotaxin-2, CKb-6), CCL25 (TECK, MIP-4a),CCL26 (eotaxin-3), CCL27 (Eskine, CTACK, ILC), CXCL1 (GROa, MGSA-a),CXCL2 (GROb, MGSA-b, MIP-2a), CXCL3 (GROg, MGSA-g, MIP-2b), CXCL4 (PF4,oncostatin A), CXCL5 (ENA-78, CXCL6 (GCP-2), CXCL7 (NAP-2, PPBP), CXCL8(IL-8, NAP-1, NAF, MDNCF), CXCL9 (Mig), CXCL10 (IP-10), CXCL11 (I-TAC),CXCL12 (SDF-1α/β), CXCL13 (BLC, BCA-1), CXCL14 (BRAK), CX3CL1(fractaline). Chemokine receptors can be CCL13 (mcp-4), CCR1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, CXCR1, XCR1 (CCXCR1).Other proteins involved in inflammation can be ABCF1, BCL6, C3, C4A,CEBPB, CRP, CARD18, IL1R1, IL1RN, CXCR2, LTB4R, and TOLLIP.

Any of the autologous compositions described herein comprising A2M canfurther comprise one or more additional non-blood derived components.Non-blood derived components can be added before, during, or afterisolation by any of the methods or using any of the systems describedherein. A non-blood derived component can be obtained from non-bloodtissues. A non-blood derived component can be an anti-coagulant. Forexample, a non-blood derived component or an anti-coagulant can be EDTA,tri-sodium citrate, water for injection (WFI), acid-citrate-dextrose(ACD), citrate-phosphate-dextrose (CPD), citrate-phosphate-doubledextrose (CP2D), citrate-phosphate-dextrose-adenine (CPDA1), or saline.

Any of the autologous compositions described herein can comprise one ormore additional blood products or blood-derived components. Bloodproducts or blood-derived components can be added before, during, orafter isolation by any of the methods described herein. Blood productsor blood-derived components can be cells, peptides, proteins, DNA, RNA,carbohydrates, or other small molecules. For example, blood products orblood-derived components can be red blood cells, white blood cells,platelets, packed red blood cells, platelet-rich plasma, plateletconcentrates, fresh plasma, fresh frozen plasma, frozen plasma,cryoprecipitate and cryosupernant.

In some embodiments, an autologous composition can contain PRP. PRP isan autologous blood product used by orthopedic healthcare providers dueto the ability of platelet concentrates to release growth factors to asurgical site along with a bone graft. Platelets can be prepared by anymeans known in the art. The cellular components of PRP productsgenerally include platelets with concentrations at least about 2 timesthe concentration of platelets in whole blood. For example, anautologous composition can contain platelets with a concentration of atleast about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, or 50 times the concentration of platelets in wholeblood. PRP products can also comprise variable concentrations of othergrowth factors released upon platelet degranulation, including, but notlimited to, transforming growth factor-beta (TGF-β), insulin-like growthfactor-1 (IGF-1), vascular endothelial growth factor (VEGF), EGF,fibroblast growth factor (FGF), and other factors. The growth factorcontent in various PRP products can vary between both patients and themethod of preparation.

In some embodiments, an autologous composition can contain platelets.Typically, platelets can be prepared by separating platelets in bloodfrom other blood components. In some embodiments, platelets can beobtained from any of the methods or systems described herein. In someembodiments, PRP can be separated or prepared from whole blood viacentrifugation. Centrifugation can be used to separate plasma andplatelets, which can be retained, from red and white blood cells, whichcan be discarded. In some embodiments, centrifugation parameters can bedesigned to achieve plasma containing approximately 70-100% of theplatelets contained in the original blood sample, and to avoid thecollection of leukocytes, such as less than 5%, for further processing.In some embodiments, concentrated platelets in blood plasma can beobtained by apheresis or pheresis (centrifugal separation during thedonor process while other components are returned to the donor) or byselective removal from whole blood after gravity or centrifugalsedimentation of blood cells.

Though PRP preparations contain growth factors, other molecularmediators are also present, including cytokines, proteases and plasmaproteins. Some of these mediators are potentially pro-inflammatory orcatabolic, and are thought to be derived from leukocytes. Though all PRPpreparations contain concentrated platelets, leukocytes and other bloodderived cells may also be present and contribute to the molecularprofile of the PRP products.

The compositions described herein can include one or more anti-oxidantsor free radical scavengers to help minimize free radical damage andpromote more rapid healing. When living tissue uses oxygen at thecellular level, unstable molecules termed free radicals are generated.Free radicals can include reactive oxygen species such as super-oxidantsand hydrogen peroxide (H202) and reactive nitrogen species such asnitric oxide and peroxynitrites. Free radicals are unstable oxygenmolecules that cause damage to cellular components such as DNA,proteins, or lipids and can induce cell death. The body naturallygenerates free radicals, which are generally neutralized in vivo bybiochemical reactions. The concentration of free radicals is extremelyhigh in a wound bed due to damage to the cells in wounded tissue. Immunecells migrating into the wound bed can induce inflammation and releasemore free radicals that do further damage to nearby cells. Inflammationcan be directly proportional to the amount of scarring that can occur.

The anti-oxidants provided in the compositions described herein caninclude, but not limited to, vitamins such as vitamin C (ascorbic acid),vitamin E, vitamin A and other retinoids; and the carotenes such as13-carotene.

In addition to its anti-oxidant functions, ascorbic acid can stimulateand organize the wound healing process due to its stimulatory effects oncollagen production. Collagen can provide strength and structure toskin, cartilage and bone, and is the scaffold upon which the regeneratedcellular and non-cellular elements of the wound can attach and grow.Ascorbic acid is known to have preservative properties, unless it isbroken down such as occurs after exposure to sunlight or another sourceof ultraviolet (UV) light. Vitamin E is also known to break down uponexposure to sunlight or UV light. As such, coverings can be used toshield the components of the compositions from UV rays upon application.

The compositions described herein can also include one or moreantiseptic, anti-infective, antibiotic, bacteriocidal, anti-fungal,anti-microbial agent or another agent that sanitizes the wound. Manywound sites are either already infected with bacteria or are susceptibleto such infection. As such it is desirable that the composition becapable of either killing bacteria or preventing the mobility or thereproduction of bacteria already present in a wound. In an embodimentthe antiseptic agent includes, but is not limited to, cetylpyridiniumchloride (CPC) and other quaternary ammonium compounds or a combinationthereof. In an embodiment, the composition includes an agent that isbacteriocidal to at least the Pseudomonas and Klebsella genera ofbacteria. The agent can be effective against E. coli, species ofStreptococcus, ShigeHa, Salmonella and most species of Staphylococcusincluding S. aureus and MRSA as well. The agent can include neosporin,vancomycin and gentamycin, and combinations thereof. The compositionsdescribed herein can include one or more plant components or plantextracts including, but not limited to, Aloe vera.

The compositions described herein can also include one or more growthfactors, cytokines or chemokines. In an embodiment, the growth factorsfor use can include PDGF, platelet-derived angiogenesis factor (PDAF),VEGF, PDEGF, platelet factor 4 (PF-4), transforming growth factor 13(TGF-13), acidic fibroblast growth factor (FGF-A), basic fibroblastgrowth factor (FGF-B), transforming growth factor a (TGF-A),insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), pthromboglobulin-related proteins (f3 TG), thrombospondin (TSP),fibronectin, von Wallebrand factor (vWF), fibropeptide A, fibrinogen,albumin, plasminogen activator inhibitor 1 (PAI-1), osteonectin,regulated upon activation normal T cell expressed and presumablysecreted (RANTES), gro-a, vitronectin, fibrin D-dimer, factor V,antithrombin ID, immunoglobulin-G (IgG), immunoglobulin-M (IgM),immunoglobulin-A (IgA), a2-macroglobulin, angiogenin, Fg-D, elastase,keratinocyte growth factor (KGF), EGF, FGF, tumor necrosis factor (TNF),FGF and interleukin-1 (IL-1), KGF-2 and combinations thereof. Each ofthese growth factors is known or believed to enhance cell or tissuegrowth. Moreover, said substances, or various combinations thereof, areknown or believed to function together in an unexpected synergisticmanner to promote wound healing. It should be appreciated that thegrowth factors can be derived from activated platelets, cultured cells,or from protein expression systems.

The compositions described herein can include concentrated platelets, orPRP as the growth factor source. The platelets can be separated from thered blood cells and white blood cells of whole blood, primarily throughdifferential centrifugation, although any suitable method for separatingplatelets from whole blood may be employed. Incidental amounts of whiteblood cells can be present due to the fact that the platelets are rarelytotally isolated from the other blood components. In an embodiment, therange of the mean platelet volume of the platelets being sequestered isin the range of about 6.6 to 8.4 femtoliters, with an average of about7.7 femtoliters. In another embodiment, the range of the mean plateletvolume of the platelets being sequestered is in the range of about250×10₁₁ per cc blood or greater. In another embodiment, the range ofthe mean platelet volume of the platelets being sequestered is in therange of about 450×10₁₁ per cc blood. The concentrated platelets or PRPcan be frozen or freeze-dried.

Some of the compositions described herein can include one or morebiochemical platelet activators or agonists including, but not limitedto, thrombin, including native thrombin, calcified thrombin, bovinethrombin (such as in Autologel), autologous thrombin, allogeneicthrombin, or recombinant human thrombin, tissue factor, von Willebrandfactor, platelet factor 4, collagen, thromboxane A2, serotonin,adenosine diphosphate (ADP), acetylcholine (ACH), or combinationsthereof to activate the platelets to release the contents of theirstored granules into the plasma. Activators and agonists can be mixedwith the plasma-containing compositions immediately prior to applicationto a patient.

In addition, the compositions described herein can include one or moreactivator co-factors including, but not limited to divalent cations suchas calcium ions, sodium ions, calcium salts in order to implement theclotting cascade and activate platelets to release the alpha granules.Suitable calcium salts include, without limitation, CaCO₃, CaSO₄, CaCl₂,CaCl₂ which can be available as calcium chloride injection, USP 10%(American Regent Laboratories, Inc., Shirley, N.Y., USA).

Systems for Production of Autologous A2M Compositions

Provided herein are systems that can be used with the methods and can beused to produce any of the compositions. A system for enrichment of A2Mfrom a sample is provided. A system can have one or more filters, acentrifuge, a pump, or a combination thereof. A system can have one ormore waste, retentate, or filtrate (used synonymously with permeate)collection modules.

One aspect of the invention is directed at a system for concentratingA2M from a fluid sample, such as a biological sample. Typically, thefluid sample is blood derived from a subject, such as a patient with achronic wound, and the system concentrates the A2M from the blood into aconcentrated A2M blood serum or concentrated A2M blood plasma. Theconcentrated sample can then be administered back to the same subject totreat the chronic wound.

The systems described herein offer a number of advantages, including lowcost and minimal parts. In some embodiments, the systems offer theadvantage of not requiring the use of a pump, such as to flow bloodthrough the system. In some embodiments, the systems provide controlover the flow of fluids through the systems beyond that offered by thepump. There is also a need for faster processing times for preparationsof therapeutic autologous blood compositions while maintaining sterilityand/or minimizing foaming of the compositions so they are suitable forinjection into a subject. Furthermore, there is a need for preparingeffective therapeutic compositions for autologous treatment usingsmaller sample sizes to minimize the amount of blood drawn from asubject Importantly, there is also a need in the art for theminimization of platelet activation and/or degranulation duringpreparation of autologous therapeutic compositions, as plateletactivation can lead to increased cytokine production and proteaseactivation, which can lead to degradation of extracellular matrixproteins, a known cause of arthritis and other inflammatory diseases.

One advantage of the blood concentrating systems and methods describedherein is that they do not require the use of a pump. Another advantageis that an operator can manually control the flow of fluids through thesystem through the use of injectors, such as syringes to move fluidsthrough the system and through the one or more filters, such as HollowFiber Tangential Flow Filters (HFTFs) or polysulfone filters of thesystems. Another advantage offered is that platelet activation anddegranulation is minimized Because the biological samples concentratedare circulated through the filters employed a minimal number of times,or not at all, platelet activation and/or degranulation can be minimizedAdditionally, because a pump is not used in some embodiments, there isno crushing of tubing or chambers of the system by a pump head, whichcan further minimize platelet activation and/or degranulation. Syringescan be used to manually move a biological sample, such as plasma, backand forth through the one or more filters of the systems. Yet anotheradvantage offered is that therapeutic autologous compositions suitablefor injection into a subject can be generated in short amounts of timeand from a minimal sample volume from a subject. The “dead space” of thesystems is minimized by removing long tubing lines, bags, and the like.The systems are also amenable to using various volumes of biologicalsamples because injectors with varying volumes can be readily used withthe systems. Additionally, biological samples separated viacentrifugation can be easily flowed into the systems, and processed. Thesystems utilize a minimal number of components, which minimizes cost aswell. As the cost is minimized, the systems can also be made disposable.Additionally, recovery of desired material can be increased using amanual system. Table 1 depicts data of recovery of various componentsfrom samples from various subjects using a manual system.

Embodiments of the invention utilize minimal mechanization toconcentrate and filter blood for autologous therapy. These lessmechanized systems offer the advantage of easy handling, less dependencyon mechanical and electrical components which may fail, and can beultimately less expensive. One embodiment of the invention, for example,uses the force of gravity and/or pressure generated manually toaccomplish concentration and filtration of a biological sample through afilter, such as a membrane, and to further process filtrate or drainwaste filtrate. Gravity and manual manipulation of positive and negativepressure applied to the system can effectuates processing of thebiological sample and washing fluid through the membrane for removingair and concentrating and filtering the biological sample.

By compartmentalization of the tangential flow filtration process, onecan produce systems that may greatly improve processing of blood,convert the system into a disposable blood concentrator or be used forpurification or isolation of certain components from biological fluidsor other fluids. Other processes and uses are also possible as willbecome apparent.

One aspect of the invention is directed at a system for concentratingA2M from a fluid sample, such as a biological sample. Typically, thefluid sample is blood derived from a subject, such as a patient withpain, and the system concentrates the A2M from the blood into aconcentrated A2M blood serum or concentrated A2M blood plasma. Theconcentrated sample can then be administered back into the same subjectto treat the pain

A system for purifying a blood component can comprise a filtrationmembrane. In some embodiments, the filtration membrane can be formed asa flat, elongated, sheet having a first surface and a second surface.The filtration membrane can have a preferred pore size to allow theselected blood components to traverse the membrane. The pore size of thefiltration membrane can simultaneously prevent the passage of otherblood components that exceed the size of the selected blood components.

TABLE 1 Trypsin Inhib A2M Fibrinogen Total Protein % Conc % of Plasma %recovery % of Plasma % recovery % % % % Patient Sample Factor Avg SD AvgSD Avg SD Avg SD Plasma Rec Plasma Rec 13-04-23 B D APIC-PRP 395 38022.0 96.3 5.6 504 105 127.6 26.5 311 79 346 88 13-04-23 J R APIC-PRP 429440 24.8 102.7 5.8 508 112 118.6 26.2 369 86 393 92 13-04-25 J HAPIC-PRP 225 148 4.2 65.6 1.9 216 17 96.0 7.5 167 74 180 80 13-04-25 F SAPIC-PRP 300 212 8.2 70.6 2.7 281 31 93.7 10.2 246 82 269 90 10-05-08 WF APIC-PRP 375 284 5.2 75.6 1.4 297 104 79.1 27.6 326 87 AVERAGEAPIC-PRP 345 293 82 361 103 273 80 303 87 SD APIC-PRP 82 119 16 136 2057 5 82 4 13-05-01 N Z Manual APIC-PRP, 400 265 5.8 66.4 1.5 296 25 73.96.3 237 59 289 72 500 kDa 13-05-03 P M Manual APIC-PRP 323 243 6.7 75.22.1 187 44 57.8 13.5 188 58 210 65 13-05-08 W F Manual APIC-PRP, 375 3055.2 81.3 1.6 272 43 72.4 11.5 302 80 500k U-shape 13-05-08 W F ManualAPIC-PRP, 300 265 8.5 88.3 2.8 249 67 83.0 22.4 265 88 500k straightAVERAGE APIC-PRP 349 270 78 251 72 213 59 266 76 SD APIC-PRP 46 26 9 4710 35 1 41 10 13-05-01 N Z Manual APIC-PRP, 400 271 2.3 67.9 0.6 292 4173.0 10.3 272 68 282 70 HPH-Mini 13-05-03 P M Manual APIC-PRP, 265 2619.5 98.6 3.6 259 30 97.9 11.3 209 79 230 87 HPH-Mini 13-05-08 W F ManualAPIC-PRP, 250 297 13.5 118.6 5.4 284 20 113.4 8.1 332 133 HPH-JuniorAVERAGE APIC-PRP 305 276 95 278 95 240 73 281 97 SD APIC-PRP 83 18 26 1720 45 8 51 32 Albumin Globulin Alk. Phos. ALT AST % % % % % % % % % %Patient Sample Plasma Rec Plasma Rec Plasma Rec Plasma Rec Plasma Rec13-04-23 B D APIC-PRP 329 83 368 93 71 18 514 130 373 95 13-04-23 J RAPIC-PRP 380 89 421 98 88 21 471 110 486 113 13-04-25 J H APIC-PRP 16272 209 93 29 13 200 89 320 142 13-04-25 F S APIC-PRP 274 91 262 87 70 23338 113 400 133 10-05-08 W F APIC-PRP 312 83 350 93 106 28 500 133 1100293 AVERAGE APIC-PRP 291 84 322 93 73 21 405 115 536 155 SD APIC-PRP 827 85 4 29 6 134 18 321 79 13-05-01 N Z Manual APIC-PRP, 295 74 278 70123 31 267 67 267 67 500 kDa 13-05-03 P M Manual APIC-PRP 210 65 210 6541 13 222 69 140 43 13-05-08 W F Manual APIC-PRP, 302 81 300 80 118 31500 133 700 187 500k U-shape 13-05-08 W F Manual APIC-PRP, 263 88 267 8971 24 450 150 400 133 500k straight AVERAGE APIC-PRP 268 77 264 76 88 25360 105 377 108 SD APIC-PRP 42 10 39 11 39 9 136 43 240 65 13-05-01 N ZManual APIC-PRP, 284 71 278 70 68 17 305 76 293 73 HPH-Mini 13-05-03 P MManual APIC-PRP, 130 87 229 86 51 19 222 84 200 76 HPH-Mini 13-05-08 W FManual APIC-PRP, 332 133 333 133 82 33 350 140 600 240 HPH-JuniorAVERAGE APIC-PRP 282 97 280 96 67 23 292 100 364 130 SD APIC-PRP 51 3252 33 16 9 65 35 209 96

An exemplary embodiment of the systems comprises a filtration modulecomprising two or more inlets and one or more filters. A volume of abiological sample flows into the filtration module and through at leastthe inlet and one or more filters of the filtration module. The flow mayalso pass through another inlet of the module after passing through thefirst inlet and one or more filters. The one or more filters can beconnected in series between the inlet and the outlet of the filtermodule. The inlets can have selectively closed valves to control flow ofthe fluid sample therein and the module may comprise multiple inlets ormultiple outlets or any combination thereof. The filtration module maybe a dead-end filtration module. Preferably, the filtration module maybe a tangential flow filtration module.

In general, a biological sample, or separated components thereof, can beapplied to the retentate side of a filter, or two or more filters inseries. A pressure gradient can be applied across the filter wherein thepressure on the retentate side of the filter is greater than thepressure on the filtrate side of the filter. The difference in pressurecan force certain blood components through the filter while other bloodcomponents, such as cells and high molecular weight molecules, which donot fit through the filter pores, can be retained. A pressure gradientcan be provided by manually injecting fluid onto a retentate side of afilter. In one embodiment, a pressure gradient can be generated byapplying a lower pressure or a vacuum, to a filtrate side of a filter.The filter of the invention can be a tangential flow filter, e.g., ahollow fiber filter. The filter of the invention can be a polysulfonefilter. In some embodiments, the filter of the invention is not apolysulfone filter. The concentrator can have an ultrafiltrationmembrane with a molecular weight cut off of 50-500. 100-500, 50, 100, or500 kDa. A pressure gradient across the membrane can be provided by amanual injector on the retentate side of the filter or from a manualinjector on the filtrate side of the filter.

In one aspect, a system can comprise a first filtration module forremoving a first blood component from a biological sample connected to asecond filtration module for removing a second blood component and/orretaining a third blood component from the biological sample afterprocessing through the first filtration module. The second filtrationmodule can also be used to remove a third blood component from thebiological sample. In some embodiments, a system can comprise a firstfiltration module comprising a first filter 30, a first channel 10(first retentate chamber), and a second channel 20 (first filtratechamber). The first channel 10 and the second channel 20 can beseparated by a first filter 30, such as a membrane. The first channel 10can comprise a first inlet end 11 and a second inlet end 12. The firstchannel 10 can have any suitable overall length. The first channel 10can have a first suitable cross-sectional area measured at the firstinlet end 11. The first channel 10 can have a second suitablecross-sectional area measured at the second inlet end 12. The firstchannel 10 can have a third suitable cross-sectional area measuredbetween the first inlet end 11 and the second inlet end 12. The secondchannel 20 (first filtrate chamber) can receive blood components thatflow within the first channel 10 (first retentate chamber) through thefirst filter 30. The first filter can have a preferred pore size chosento allow the selected blood components, such as proteins, to traversethe membrane. The pore size of the first filter 30 can prevent thepassage of other blood components, such as cells, that exceed the sizeof the selected blood components.

In some embodiments, a hollow fiber filter is used to separate the firstchannel 10 (first retentate chamber) and the second channel 20 (firstfiltrate chamber). In a preferred embodiment, the first filtrate chamber20 can at least partially enclose the first retentate chamber 10. Atleast a portion of the first retentate chamber 10 wall can be the firstmembrane. The first filtrate chamber 20 can comprise an inner wall andan outer wall. At least a portion of the inner wall can be the firstmembrane portion 30 of the first retentate chamber 10 wall. The firstfiltrate chamber 20 outer wall can comprise an outer barrier impermeableto fluid. The first membrane portion 30 can comprise a hollow fiberfilter, which can comprise multiple hollow fibers that run the length ofthe first channel 10 and/or second channel 20 in parallel. Hollow fiberscan be embedded at each end of the channels. The hollow fiber's lumenscan be retained open and can form a continuous passage through each ofthe lumens from one end of the channel to the other. The hollow fiberscan be enclosed by the outer wall of the channels in the filtrationmodules. As a result, there can be a chamber bounded by the secondchannel 20 wall and the outer walls of the hollow fibers. That chambercan be used as the first filtrate chamber 20. The internal spaces of thehollow fibers can constitute part of the first retentate chamber 10 inthe system described herein. The outer walls of the channels can haveone or more ports from which the filtrate can be collected, flowed,and/or removed.

The first channel can be extended further than the internal spaces ofthe hollow fibers by a first adaptor 41 and/or a second adaptor 42 thatfit to the first inlet 11 and second inlet 12, respectively, of thefirst channel 10. One or more adapters in conjunction with an inlet ofthe channel can be part of the retentate chamber. One or more adaptersin conjunction with an inlet of the channel can be in fluid connectionwith the retentate chamber. Depending on the direction of fluid flowthrough the fibers, the retentate channel can collect fluid as it exitsthe hollow fibers Depending on the direction of fluid flow through thefibers, the retentate channel can allow fluid arriving through an inletto interface with the hollow fiber's open ends and distribute throughthe fibers and flow towards the other end of the channel Each adaptercan comprise a first and a second end. The first end can be fitted tothe channel and the second end can comprise an opening connectable to aninjector, vessel, or a pump. In one embodiment, the injector can beconnected to the adapter via a line to allow for fluid flow. In oneembodiment, the injector can be connected directly to the adapter. Insome embodiments, the adapter may form part of the injector where partor the entire content of the injector may be contained within theadapter. The adapter can be connected directly to a manual injector but,if desired, a pump may be connected to the adaptor. When a connectingline is added to an adapter, the retentate chamber can include the spaceinside the connecting line. When a connecting line is connected at oneend to an adapter and at its other end to a vessel, the interior of thevessel can be included as part of the first retentate chamber 10.

A first injector 51 can be connected to the first inlet end 11 of thefirst channel 10, optionally through an adaptor. A second injector 52can be connected to the second inlet 12 of the first channel 10,optionally through an adaptor. The first injector 51 and/or the secondinjector 52 can hold, collect, or be used to inject fluid at variousstages of the systems operation. The first injector 51 can comprise abiological sample, such as blood. Activation of the first injector 51can cause the biological sample to flow through the first inlet 11,through the first channel 10 and over the first membrane 30. Theinjected biological sample can flow through the second inlet 12 into thesecond injector 52. Activation of the second injector 52 can cause thebiological sample to flow back through the second inlet 12, through thefirst channel 10 and over the first membrane 30. The biological samplecan flow back through the first inlet 11 and back into the firstinjector 51. This process can be repeated until a sufficient amount ofthe filtered blood sample has passed through the first filter 30 andinto the second channel 20.

In some embodiments, the system comprises a first filtrate reservoir 75connected to the first filtrate chamber 20. Fluid can flow between thefirst filtrate reservoir 75 and the first filtrate chamber 20. Thereservoir can enclose the portion of the first filtrate chamber 20 thatopens into the first filtrate reservoir 75 (e.g., a port). In someembodiments, the system comprises a second filtration module connectedto the first filtrate reservoir 75. Flow of fluid from the firstfiltrate chamber 20 to the filtrate reservoir 75 and flow of fluidbetween the filtrate reservoir 75 and the second filtration module canbe closed, open, or controlled through the use of a valve, such as astop cock.

The second channel 20 can have any suitable overall length. The secondchannel 20 can have a cross-sectional area. The second channel 20 canhave a port 70 connected thereto. The port 70 can be optionally fluidlyconnected to a third inlet 71 of a third channel 72. The third channel72 can have any suitable overall length. The port 70 can be optionallyfluidly connected to a collection vessel 300. The collection vessel 300can be the third injector 53 or the fourth injector 54. The collectionvessel 300 can be adapted to fit a first inlet end 11 and/or a secondinlet end 12. The collection vessel 300 can be adapted to fit a firstadaptor 41 and/or a second adaptor 42. The collection vessel 300 can bea syringe.

The third channel 72 can have a suitable cross-sectional area. In someembodiments, the third channel can be arranged substantiallyperpendicular to the first channel 10, second channel 20, or both. Thethird channel 72 can comprise a first outlet 74 connected to a vessel 75(e.g., a filtrate reservoir). The third channel 72 can comprise a valve76 situated between the first outlet 74 and third inlet 71 of the thirdchannel. A fourth channel 80 can be connected to the valve 76 via afourth inlet 81.

Blood components that flow from the first channel 10 through the firstfilter 30 into the second channel 20 and through the port 70 can flowthrough the third inlet of the third channel 71, the third channel 72,and the first outlet of the third channel 74 into the vessel 75 when thevalve 76 is in a first open configuration. The fourth channel 80 can bein fluid connection with a third injector 53. In some embodiments, whenthe valve 76 is in the first open configuration, fluid is prevented fromflowing into the fourth channel 80.

Blood components in the vessel 80 can flow through the third channel 72and through the fourth channel 80 when the valve 76 is in a second openconfiguration. In some embodiments, this can be caused by activation ofthe third injector 53. The second open configuration can prevent flow ofblood components from the vessel 75 through a portion of the thirdchannel 72 upstream of the valve 76.

The system can have a fifth channel 90 (second retentate chamber) and asixth channel 100 (second filtrate chamber). The fourth channel 80 canbe connected to a fifth channel 90. The fifth channel can be connectedto a third injector 53, such as through an adaptor 43. The fifth channel90 and the sixth channel 100 can be separated by a second filter 110,such as a membrane. The fifth channel 90 can comprise a fifth inlet 13and a sixth inlet 14.

A third injector 53 can be connected to the fifth inlet end 13, such asthrough an adaptor 43. A fourth injector 54 can be connected to thesixth inlet 14, such as through an adaptor 44. The third injector 53and/or fourth injector 54 can hold, collect, or be used to inject fluidat various stages of the system's operation. The third injector 53 cancomprise the filtered biological sample from the vessel 75. Activationof the third injector 53 can cause the filtered biological sample toflow through the fifth inlet 13, through the fifth channel 90 and overthe second membrane 110. The injected biological sample can flow throughthe sixth inlet 14 into the fourth injector 54. Activation of the fourthinjector 54 can cause the biological sample to flow back through thesixth inlet 14, through the fifth channel 90 and over the secondmembrane 110. The biological sample can flow back through the fifthinlet 13 and back into the third injector 53. This process can berepeated until a sufficient amount of the filtered blood sample haspassed through the second filter 110 and into the sixth channel 100.This process can result in the formation of a concentrated biologicalsample in the fifth channel 90. The concentrated biological sample canbe collected in the third injector 53 or the fourth injector 54.

The fifth channel 90 can have any suitable overall length. The fifthchannel 90 can have a first cross-sectional area measured at the fifthinlet 13. The fifth channel 90 can have a second cross-sectional areameasured at the sixth inlet 14. The fifth channel 90 can have a thirdcross-sectional area measured between the fifth inlet 13 and the sixthinlet 14. The sixth channel 100 can receive blood components that flowfrom the fifth channel 90 through the second filter 110. The secondfilter 110 can have a preferred pore size chosen to prevent desiredblood components, such as high molecular weight proteins, to traversethe membrane. The pore size of the second filter 110 can simultaneouslyallow the passage of other blood components, such as smaller molecularweight proteins that do not exceed the size of the desired bloodcomponents into the sixth channel 100.

The sixth channel 100 can have any suitable overall length. The sixthchannel 100 can have a suitable cross-sectional area. The sixth channel100 can have a port 120 connected thereto. The port 120 can be fluidlyconnected to a seventh inlet of a seventh channel 121. The seventhchannel 122 can have any suitable overall length. The seventh channel122 can have a suitable cross-sectional area. In some embodiments, theseventh channel can be arranged substantially perpendicular to the fifthchannel 90, sixth channel 100, or both. The seventh channel 122 cancomprise a second outlet 123 connected to a second vessel 124. Bloodcomponents that flow from the fifth channel 90 through the second filter110 into the sixth channel 100 and through the second port 120 can flowthrough the seventh inlet of the seventh channel 121, the seventhchannel 122, and the outlet of the seventh channel 123 into the vessel124. In some embodiments, the seventh vessel 124 is a waste collectionvessel.

In a preferred embodiment, a hollow fiber filter is used to separate thefifth channel 90 (second retentate chamber) and the sixth channel 100(second filtrate chamber). In a preferred embodiment, the secondfiltrate chamber 100 can at least partially enclose the second retentatechamber 90. At least a portion of the second retentate chamber 90 wallcan be the second membrane portion 110. The second filtrate chamber 100can comprise an inner wall and an outer wall. At least a portion of theinner wall can be the second membrane portion 100 of the second filtratechamber 90 wall. The second filtrate chamber 90 outer wall can comprisean outer barrier impermeable to fluid. The second membrane 110 portioncan comprise a hollow fiber filter, which can comprise multiple hollowfibers that run the length of the fifth channel 90 and/or sixth channel100 in parallel. Hollow fibers can be embedded at each end of thechannels. The hollow fiber's lumens can be retained open and can form acontinuous passage through each of the lumens from one end of thechannel to the other. The hollow fibers can be enclosed by the outerwall of the channels in the filtration modules. As a result, there canbe a chamber bounded by the sixth channel 100 wall and the outer wallsof the hollow fibers. That chamber can be used as the second filtratechamber 100. The internal spaces of the hollow fibers can constitutepart of the second retentate chamber 90 in the system described herein.The outer walls of the channels can have one or more ports from whichfiltrate can be collected, flowed, and/or removed.

The fifth channel 90 can be extended further than the internal spaces ofthe hollow fibers by a third adapter 43 and/or fourth adapter 44 thatfit to the third inlet 13 and/or fourth inlet 14 of the fifth channel90, respectively. Each adapter in conjunction with an inlet of thechannel can be part of the second retentate chamber 90. Depending on thedirection of fluid flow through the fibers, the second retentate channel90 can collect fluid as it exits the hollow fibers and/or allow fluidarriving through an inlet to interface with the hollow fiber's open endsand distribute itself among those hollow fibers for purposes ofcontinuing its path towards the other end of the channel. Each adaptercan comprise a first and a second end. The first end can be fitted tothe channel 90 and the second end can comprise an opening connectable toan injector, vessel, or a pump. In one embodiment, the injector can beconnected to the adapter via a line to allow for fluid flow but. In oneembodiment, the injector can be connected directly to the adapter. Insome embodiments, the adapter may form part of the injector where partor the entire content of the injector may be contained within theadapter. Normally the adapter is connected directly to a manual injectorbut, if desired, a pump mat be connected to the adaptor. When aconnecting line is added to an adapter, the second retentate chamber 90can include the space inside the connecting line. When a connecting lineis connected at one end to an adapter and at its other end to a vessel,the interior of the vessel can be included as part of the secondretentate chamber 90.

In some embodiments, a sample that flows through the first filter 30 canflow through port 70 and into a collection vessel 300. The collectionvessel 300 can be the third injector 53 or the fourth injector 54. Thecollection vessel 300 can be adapted to fit a first inlet end 11 and/ora second inlet end 12. The collection vessel 300 can be adapted to fit afirst adaptor 41 and/or a second adaptor 42. The collection vessel 300can be a syringe. The collection vessel 300 can then be removed from theport, such as after collecting the first filtrate, and the collectionvessel 300 can be connected to the first inlet end 11 or a second inletend 12. The collected sample can then be flowed from the collectionvessel 300 through the first inlet end 11 or a second inlet end 12 andinto the first retentate chamber 10 and processed as described above. Insuch embodiments, there is no need for the second filtrate chamber 100and first retentate chamber 10 to be in fluid connection.

In another aspect, a system can comprise a centrifuge and a device forremoving a first blood component and/or retaining a second bloodcomponent from a portion of a biological sample after centrifugation. Insome embodiments, a system can comprise a first filtration module forremoving a first blood component from a portion of a centrifugedbiological sample connected to a second filtration module for removing asecond blood component and/or retaining a third blood component from aportion of a centrifuged biological sample after processing through thefirst filtration module. The second filtration module can also be usedto remove a third blood component from a portion of a centrifugedbiological sample. In some embodiments, a system can comprise a firstfiltration module comprising a first filter 230, a first channel 210(first retentate chamber), and a second channel 220 (first filtratechamber). The first channel 210 and the second channel 220 can beseparated by a first filter 230, such as a membrane. The first channel210 can comprise a first inlet end 211 and a second inlet end 212. Thefirst channel 210 can have any suitable overall length. The firstchannel 210 can have a first suitable cross-sectional area measured atthe first inlet end 211. The first channel 210 can have a secondsuitable cross-sectional area measured at the second inlet end 212. Thefirst channel 210 can have a third suitable cross-sectional areameasured between the first inlet end 211 and the second inlet end 212.The second channel 220 (first filtrate chamber) can receive bloodcomponents that flow within the first channel 210 (first retentatechamber) through the first filter 230. The first filter can have apreferred pore size chosen to allow the selected blood components, suchas proteins, to traverse the membrane. The pore size of the first filter230 can prevent the passage of other blood components, such as cells,that exceed the size of the selected blood components.

In a preferred embodiment, a hollow fiber filter is used to separate thefirst channel 210 (first retentate chamber) and the second channel 220(first filtrate chamber). In a preferred embodiment, the first filtratechamber 220 can at least partially enclose the first retentate chamber210. At least a portion of the first retentate chamber 210 wall can bethe first membrane portion 230. The first filtrate chamber 220 cancomprise an inner wall and an outer wall. At least a portion of theinner wall can be the first membrane portion 230 of the first retentatechamber 210 wall. The first filtrate chamber 220 outer wall can comprisean outer barrier impermeable to fluid. The first membrane portion 230can comprise a hollow fiber filter, which can comprise multiple hollowfibers that run the length of the first channel 210 and/or secondchannel 220 in parallel. Hollow fibers can be embedded at each end ofthe channels. The hollow fiber's lumens can be retained open and canform a continuous passage through each of the lumens from one end of thechannel to the other. The hollow fibers can be enclosed by the outerwall of the channels in the filtration modules. As a result, there canbe a chamber bounded by the second channel 220 wall and the outer wallsof the hollow fibers. That chamber can be used as the first filtratechamber 220. The internal spaces of the hollow fibers can constitutepart of the first retentate chamber 210 in the system described herein.The outer walls of the channels can have one or more ports from whichthe filtrate can be collected, flowed, and/or removed.

The first channel can be extended further than the internal spaces ofthe hollow fibers by a first adaptor 241 and/or a second adaptor 242that fit to the first inlet 211 and second inlet 212 respectively of thefirst channel 210. Each adapter in conjunction with an inlet of thechannel can be part of the retentate chamber. Depending on the directionof fluid flow through the fibers, the retentate channel can collectfluid as it exits the hollow fibers and/or allow fluid arriving throughan inlet to interface with the hollow fiber's open ends and distributeitself among those hollow fibers for purposes of continuing its pathtowards the other end of the channel Each adapter can comprise a firstand a second end. The first end can be fitted to the channel and thesecond end can comprise an opening connectable to an injector, vessel,or a pump. In one embodiment, the injector can be connected to theadapter via a line to allow for fluid flow but. In one embodiment, theinjector can be connected directly to the adapter. In some embodiments,the adapter may form part of the injector where part or the entirecontent of the injector may be contained within the adapter. Normallythe adapter is connected directly to a manual injector but, if desired,a pump mat be connected to the adaptor. When a connecting line is addedto an adapter, the retentate chamber can include the space inside theconnecting line. When a connecting line is connected at one end to anadapter and at its other end to a vessel, the interior of the vessel canbe included as part of the first retentate chamber 210.

A first injector 251 can be connected to the first inlet end 211 of thefirst channel 210, optionally through an adaptor. A second injector 252can be connected to the second inlet 212 of the first channel 210,optionally through an adaptor. The first injector 251 and/or the secondinjector 252 can hold, collect, or be used to inject fluid at variousstages of the systems operation. The first injector 251 can comprise abiological sample, such as blood. Activation of the first injector 251can cause the biological sample to flow through the first inlet 211,through the first channel 210 and over the first membrane 230. Theinjected biological sample can flow through the second inlet 212 intothe second injector 252. Activation of the second injector 252 can causethe biological sample to flow back through the second inlet 212, throughthe first channel 210 and over the first membrane 230. The biologicalsample can flow back through the first inlet 211 and back into the firstinjector 251. This process can be repeated until a sufficient amount ofthe filtered blood sample has passed through the first filter 230 andinto the second channel 220.

In some embodiments, the system comprises a first filtrate reservoir 275connected to the first filtrate chamber 220. Fluid can flow between thefirst filtrate reservoir 275 and the first filtrate chamber 220. Thereservoir can enclose the portion of the first filtrate chamber 220 thatopens into the first filtrate reservoir 275 (e.g., a port). In someembodiments, the system comprises a second filtration module connectedto the first filtrate reservoir 275. Flow of fluid from the firstfiltrate chamber 220 to the filtrate reservoir 275 and flow of fluidbetween the filtrate reservoir 275 and the second filtration module canbe closed, open, or controlled through the use of a valve, such as astop cock.

The second channel 220 can have any suitable overall length. The secondchannel 220 can have a cross-sectional area. The second channel 220 canhave a port 270 connected thereto. The port 270 can be fluidly connectedto a third inlet 271 of a third channel 272. The third channel 272 canhave any suitable overall length. The third channel 272 can have asuitable cross-sectional area. In some embodiments, the third channelcan be arranged substantially perpendicular to the first channel 210,second channel 220, or both. The third channel 272 can comprise a firstoutlet 274 connected to a vessel 275 (e.g., a filtrate reservoir).

Blood components that flow from the first channel 210 through the firstfilter 230 into the second channel 220 and through the port 270 can flowthrough the third inlet of the third channel 271, the third channel 272,and the first outlet of the third channel 274 into the vessel 275 whenthe system is in use. In some embodiments, the vessel 275 is a wastecollection vessel

Any of the injectors described herein can be used for injecting abiological sample into the systems described herein. Any of theinjectors described herein can be used to apply pressure to the systemsdescribed herein and to cause fluid to flow through the system. Any ofthe injectors described herein can be manually operated. Exemplaryinjectors include syringes. Any of the injectors described herein canalso function as sample collection modules as the sample passes throughvarious stages of the systems.

In some embodiments, a system can comprise one or more Luer-lockadapters. In some embodiments a Luer lock adapter can be capped orclosed. In some embodiments, a system can comprise one or more dead-endfilters. In some embodiments, one or more of the inlets can be closed orsealed.

In some embodiments, the sample can be processed through the systemmanually. For example, a sample can be processed through the systemwithout the use of a pump. In some embodiments, the sample can beprocessed through the system by pushing or pulling the sample throughthe system using a pressure generating device, such as a syringe.Injectors can also be used as flow control devices to control the flowof the biological sample through one or more portions of the systemsdescribed herein. In some embodiments, other suitable flow controldevices include actuators, clamps, ball valves, diaphragm valves, needlevalves, and pumps. Flow control devices can be variable flow, i.e.,capable of providing varying flow rates, and binary on/off that open topermit flow and close to block flow. Suitable pumps include, but are notlimited to, vane pumps, tubing pumps, rotary pumps, and diaphragm pumps,and may be chosen based upon, for example, the desired flow rate.

In any of the systems described herein, a filtration membrane ispositioned between, and separates, a retentate chamber and a filtratechamber. Many configurations are possible for the retentate chamber andthe filtrate chamber. For example the retentate chamber and the filtratechamber can be configured in a side-by-side relationship. Alternatively,the retentate chamber and filtrate chamber can be configured to becoaxially disposed with either the retentate chamber or filtrate chamberpositioned inside of the other. Any configuration of the retentatechamber and filtrate chamber that forces the biological sample to flowtangentially over opposite sides of a filtration membrane can be adaptedfor use in the present invention.

Changing pressure gradients and both axial flow and perpendicular flowto the membrane surface occur in the alternating tangential flowprocess. Upon activation of the injector on one side of the retentatechamber, the pressure in the injector is greater than the pressure inthe retentate chamber. The retentate flows forward from the injector,i.e., through the retentate chamber and over the filter element. Also,some of the liquid is forced across the filter membrane into thefiltrate chamber. When the filtrate chamber is enclosed, the entry offiltrate can pressurize the filtrate chamber. Conversely, uponactivation of the injector on the opposite side of the retentatechamber, the pressure in the injector is greater than the pressure inthe retentate chamber, so that sample flows in reverse, from theinjector on one side of the retentate chamber to the injector on theother side of the retentate chamber. This reversal in flow maintains themembrane and inhibits clogging. This effect is further enhanced byanother kind of transmembrane flow, one which forms when the resistanceto axial flow inside the hollow fiber, or 1 μmen side, is greater thanin the external, shell, side of the hollow fiber. Therefore, during theflow of the sample onto the filter, pressurized sample is forced intothe hollow fibers and will take the path of least resistance and thesample will not only flow through the lumens, but also across themembrane, into the filtrate chamber. An axial pressure gradient forms onboth sides of the filter causing fluid flow towards the exit end of thefilter. As a result, the sample flow has the capacity to exchange fluidsfrom the retentate chamber to the filtrate chamber. Such exchange can behighly beneficial for processing biological samples for generation ofthe composition described herein.

Another aspect of the invention reduces, eliminates, or substantiallyeliminates the introduction of air and/or generation of foam in thecompositions produced using the methods or systems described herein.

Some of the key features of the system can utilize a fluid, such assterile saline, to load the filter and purge air out of the systems andtheir various components before introduction and processing of thebiological sample. This purging step can substantially eliminateintroduction of air or bubbles (foaming) into the biological sampleduring the filtration and concentration process. Thus, the systems andmethods are amenable to generate therapeutic compositions that can bereadily administered into the blood stream of a subject.

In some embodiments, a pressure differential across a filter is notachieved by a vacuum applied to the system. In some embodiments, asystem does not comprise a stopcock. In some embodiments, a system doesnot comprise an adaptor to an injector with a Luer lock fitting.

In some embodiments, the systems do not produce a composition comprisingfibrinogen or concentrated fibrinogen. In some embodiments, acomposition does not comprise fibrinogen or concentrated fibrinogen,such as at least about 1.1, 1.5, 2, 3, 4, 5, 10, or 20 fold concentratedfibrinogen. In some embodiments, a composition comprises concentratedfibrinogen at most about 1.1, 1.5, 2, 3, 4, 5, 10, or 20 foldconcentrated fibrinogen. In some embodiments, the systems do not producea composition comprising clotting factors or concentrated clottingfactors such as Factor V and Factor X, and other undesired constituents,such as growth factors. In some embodiments, a composition does notcomprise clotting factors or concentrated clotting factors such asFactor V and Factor X, and other undesired constituents, such as growthfactors.

Injectors

Any of the injectors described herein can be used for injecting abiological sample into the systems described herein. Any of theinjectors described herein can be used to apply pressure to the systemsdescribed herein and to cause fluid to flow through the system. Any ofthe injectors described herein can be manually operated. Exemplaryinjectors include syringes. Any of the injectors described herein canalso function as sample collection modules as the sample passes throughvarious stages of the systems.

According to one embodiment, the injector comprises a syringe. A typicalsyringe comprises a generally cylindrical barrel having opposed proximaland distal ends with at least one chamber formed between the ends forreceiving a substance such as a biological sample. A plunger istypically sealably disposed within the barrel and movable with respectthereto, and sealing means may be sealably disposed approximate to thedistal end of the barrel. A syringe can include an elongate barrel orcylinder having an open, proximal end and a distal end with at least onehollow chamber formed between the proximal and distal ends for receivinga biological sample. A plunger may be situated at the open, proximalend. A plunger can be moved by means of a plunger rod, which can besecured to the plunger, for example, by screwing or gluing. At the sameend where the plunger is situated, the barrel may have a finger grip,which is secured to the barrel. A finger grip preferably consists ofslightly resilient material, for example plastics. In anotherembodiment, the finger grip is a flange-like part of the barrelprojecting radially outwards. Of course, other constructions known tothose skilled in the art are possible. A stopper, which closes thebarrel, may be situated in the end of the barrel remote from theplunger. The plunger and the stopper are preferably manufactured from anelastic material and, most preferably, from rubber of a pharmaceuticalquality.

In preferred embodiments, an injection needle is not secured to thebarrel. In further embodiments, the syringe is not stored with a needlein position, e.g., a needleless syringe. Although the syringe barrel caninclude a locking Luer-type collar, it is within the purview of thepresent invention to include syringe barrels without a collar, syringebarrels having an eccentrically positioned nozzle and various othernozzle-like structures. It is only required that there is an aperture onthe distal end of the syringe barrel in fluid communication with theinterior of the syringe barrel.

Vessels

Any of the reservoirs described herein can be any suitable vessel.Suitable vessels include any collection device including, but notlimited to, tubes such as test tubes and centrifuge tubes; closed systemblood collection devices, such as collection bags; syringes, catheters,such as central lines; microtiter and other multi-well plates; arrays;tubing; laboratory vessels such as flasks, spinner flasks, rollerbottles, vials, microscope slides, microscope slide assemblies,coverslips, films and porous substrates and assemblies; pipettes andpipette tips, etc.; tissue and other biological sample collectioncontainers; and any other container suitable for holding a biologicalsample, as well as containers and elements involved in transferringsamples. In one aspect of the invention, a sample collection tube havinga separating member (e.g., a mechanical separating element, a gel or afilter mechanism) for separating blood components is used. The vesselmay also comprise a collection bag suitable for holding a biologicalsample such as, for example, a blood collecting bag, a blood plasma bag,a buffy coat bag, a platelet bag or the like.

Plastic or glass can be used to manufacture the reservoirs and vesselsused in the present invention. Some preferred materials used tomanufacture the vessels include polypropylene, polyethylene,polyethyleneterephthalate, polystyrene, polycarbonate and cellulosics.More expensive plastics such as polytetrafluoroethylene and otherfluorinated polymers may also be used. In addition to the materialsmentioned above, examples of other suitable materials for the vesselsused in the present invention include polyolefins, polyamides,polyesters, silicones, polyurethanes, epoxies, acrylics, polyacrylates,polysulfones, polymethacrylates, PEEK, polyimide and fluoropolymers suchas PTFE Teflon®, FEP Teflon®, Tefzel®, poly(vinylidene fluoride), PVDFand perfluoroalkoxy resins. Glass products including silica glass arealso used to manufacture the vessels. One exemplary glass product isPYREX® (available from Coming Glass, Corning, N.Y.). Ceramic collectiondevices can be used according to embodiments of the invention.Cellulosic products such as paper and reinforced paper containers canalso be used to form vessels according to the invention.

Filters/Membranes

TFF is a filtration process whereby the solution is constantly flowingover the membrane to prevent pores from becoming clogged by cells andproteins. As used in the systems and methods described herein, TFF candiscourage unwanted particles, cells and/or other large proteins fromblocking the membrane pores and allowing the flow of small molecules andproteins. The use of hollow fiber membranes can increase the surfacearea that is available for filtration. The 50, 100, or 500 kDa molecularweight cutoff of the membrane permits small molecules and proteins suchas cytokines, chemokines, and proteases to pass through, eventuallyleading to waste, but retaining larger particles (>50, 100, or 500 kDa)such as platelets.

In some embodiments the one or more filters of the filtration module(s)comprise at least a first and a second filter. The first filter screensout cells, particles, and other molecules larger than 1 or 2 microns.The second filter screens out molecules having a weight less than about50, 100, or 500 kDa. The second filter may also retain molecules havinga weight of more than about 50, 100, or 500 kDa. In some embodiments thefirst and the second filters comprise cross-flow filters, such as hollowfiber filters.

Some embodiments of the invention further comprise a pump adapted to befluidly coupled with the filtration module either upstream of the inletof the filtration module or downstream of the outlet of the filtrationmodule. The pump is further adapted to produce a flow of the fluidsample that passes through the one or more filters of the filter module.In some embodiments of the invention, the filter module furthercomprises at least one reservoir.

In an exemplary embodiment of the invention wherein the first filtercomprises a cross-flow filter that screens out cells particles and othermolecules lager than 1 or 2 microns. A retentate of this filtercontaining the cells, particles, and other molecules larger than 1 or 2microns of the fluid sample is stored in a first retentate reservoir.Alternatively, the retentate of the first filter is discarded. Afiltrate of the first filter is directed to a first filtrate reservoir,the first filtrate reservoir is then typically connected to the secondfilter. The filtrate of the first filter flows through the secondfilter. The second filter may typically be a cross flow filter adaptedto retain molecules of weight more than about 50, 100, or 500 kDa. Aretentate of the second filter comprises these molecules of weight morethan about 50, 100, or 500 kDa may be retained in the first filtratecollection reservoir. The retentate of the second filter typicallycomprises the concentrated A2M of the fluid sample. A filtrate of thesecond filter can be directed to a separate second filter filtratereservoir. Alternatively, the filtrate of the second reservoir may beredirected through the outlet of the filtration module and circulatedback to the inlet of the filtration module such that the fluid sample isprocessed by the filtration module multiple times or continuously.

In some embodiments a system further comprises a centrifuge. A fluidsample can be centrifuged to produce a supernatant and a pellet, thesupernatant containing small molecules and A2M but not large particlessuch as cells. The pellet contains the large particles such as cellspresent in the fluid sample. The supernatant can then be directedthrough a filtration module. The filtration module can comprise at leastone filter adapted to retain molecules of weight more than about 50,100, or 500 kDa. The at least one filter typically can comprise a 50,100, or 500 kDa cross flow filter as describe above. The retentate ofthe 50, 100, or 500 kDa cross flow filter comprising the A2M of thesupernatant can be retained in a retentate reservoir. The filtrate ofthe at least one filter can be directed to a waste reservoir ordiscarded. Alternatively, the filtrate of the at least one filter may bedirected to the filter module outlet where can be redirected to thefilter module inlet such that the supernatant of the fluid sample passesthrough the filter module on or more times.

A system can comprise a filtration module. The filtration module cancomprise an inlet, an outlet and one or more filters fluidly connectedin series between the inlet and the outlet. The filtration module can beenclosed in a housing, such as a column or jacket. A method employing asystem can comprise removing cells from a sample. The method can furthercomprise manually pushing the fluid sample through the filtration moduleinlet and the one or more filters to produce a concentrated A2M serum orplasma. The fluid sample may also be manually pushed through the outlet.Pushing the fluid sample can be accomplished via use of an injector,such as a syringe, fluidly connected to the filtration module upstreamof the inlet and/or downstream of the outlet. The one or more filters ofthe filtration module can comprise at least one 50, 100, or 500 kDafilter configured to retain molecules with a weight more than about 50,100, or 500 kDa.

In some embodiments, removing cells from the fluid sample can compriseproviding a centrifuge, centrifuging the fluid sample, and obtaining aresultant supernatant of the fluid sample. A resultant pellet of thefluid sample comprising cells and large molecules may be retained. Theresultant supernatant of the fluid sample typically comprises A2M andsmall molecules but not cells and large molecules. The supernatant ofthe fluid sample can then be manually flowed through the filtrationmodule to concentrate the A2M. The 50, 100, or 500 kDa filter of thefiltration module can be a 50, 100, or 500 kDa cross-flow filter; aretentate of the 50, 100, or 500 kDa cross-flow filter can be retainedand can comprise a concentrated A2M serum.

In some embodiments, removing cells from the fluid sample comprisesfiltering the fluid sample with a first filter of the filtration moduleadapted to screen out cells, particles, and molecules larger than atleast about 1 or 2 microns, for example, at least about 3, 4, 5, 6, 7,8, 9, or 10 microns. The first filter can be a cross-flow filter and afiltrate of the first filter can be directed to the at least one 50,100, or 500 kDa filter of the filtration module. The filtrate of thefirst filter may be stored in a first filtrate reservoir. The 500 kDafilter of the filtration module can be a 50, 100, or 500 kDa cross-flowfilter; a retentate of the 500 kDa cross-flow filter can be retained inthe first filtrate reservoir and can comprise the concentrated A2Mserum. A filtrate of the 50, 100, or 500 kDa cross flow filter may bestored, discarded, or directed to the inlet of the filtration modulesuch that it can be further filtered.

A system can comprise one or more filters with a molecular weightcut-off of at most about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa,35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa,80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa,160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa,240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa,320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa,400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa,480 kDa, 490 kDa or lower to obtain one or more filtrates andretentates, such as an A2M enriched retentate or A2M concentratedretentate.

A system can comprise one or more filters with a pore size of at mostabout 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6μm, 7 μm, 8 μm, 9 μm, or 10 μm, for example, to remove or collect cellsand/or particles.

It should be understood that features of the above described method andsystem embodiments may be combined and interchanged with one another.

Cells and particles with a size of 0.6 μm or more, and other moleculeswith a molecular weight of 50, 100, or 500 kDa or less can be removedfrom the sample by flowing or passing a sample through one or morefilters contained within the system in sequence. Removed cells andparticles can be disposed of or collected in a waste module. Forexample, cells and particles of 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2μm, or 3 μm or more, and other molecules with a molecular weight of 5kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 90 kDa, 100 kDa,110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa,190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa,270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa,350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa,430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, or lesscan be removed by the one or more filters of the systems and can befurther processed or deposited into one or more waste modules. A samplecan be flowed through one or more filters by applying a pressuremanually, such as by pushing and or pulling an injector of the system.In some embodiments, a sample can be flowed through one or more filtersby applying centrifugal force using a centrifuge of the system, using apump of the system, or a combination thereof. A system can furthercomprise a collection module or reservoir. A retentate or filtrate canbe collected or isolated in the collection module, for example aretentate with an A2M enriched sample can be isolated in a collectionmodule, after passing a sample through one or more filters. A system canfurther comprise a sample loading module. A sample loading module can beoperable to introduce the sample into the system. A sample loadingmodule can be directly or indirectly attached to the blood stream of asubject.

The first step in the system can be either a centrifugation step orfiltration step. The collected blood can be centrifuged at a particularcentrifugal force that allows the precipitation of the red blood cellsand white blood cells and other particles and debris, allowing plasmaproteins and platelets in the supernatant. This process can also beachieved by filtration using a hollow fiber membrane that will allow theplasma proteins and platelets to go through the membrane into thefiltrate and prevent the red blood cells and white blood cells and otherparticles and debris to remain in the retentate. In some embodiments,the supernatant from the centrifugation step or the filtrate from thefiltration step can be filtered on the second filter where proteins 50,100, or 500 kDa or larger can be retained by the filter and smallerproteins than 50, 100, or 500 kDa and other molecules can pass throughthe membrane into the filtrate. The concentrated retentate can then becollected and injected into the patient.

In some embodiments, a collection receptacle with platelets and plasmacan be connected to any of the systems described herein, such as withhematologic tubing, and manually passed through a filter, such as hollowfiber tangential flow filter (HFTFF), that uses a molecular weightcutoff membrane, such as a 50, 100, or 500 kDa molecular weight cutoffmembrane, of modified polyethersulfone. In some embodiments, a pump,such as a peristaltic pump, can be used. In some embodiments, theflow-through port of the filter can be connected using hematologictubing back to the collection bottle in a closed-loop. In someembodiments, the filtrate port of the filter can also be connected usingtubing connected to waste. In some embodiments, no priming of theflow-circuit is necessary. In preferred embodiments, priming of theflow-circuit is accomplished by flowing a fluid through the system toremove air from the system. In some embodiments, priming of a systemsubstantially prevents foaming of the compositions administeredtherapeutically, such as to a joint.

As a non-limiting example, a system, such as an APIC system (AutologousPlatelet Integrated Concentrate system) can be run until the plasmareaches between 2-10 times the concentrations as found in whole blood.For example, a system can be run until the plasma reaches between 2-10,1-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 1-9, 3-8, 3-7, 3-6, 3-5, 3-4,4-10, 1-9, 4-8, 4-7, 4-6, 4-5, 5-10, 1-9, 5-8, 5-7, 5-6, 6-10, 1-9, 6-8,6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times the concentrations asfound in whole blood. In some embodiments, the starting volume of thebiological sample injected in to the systems described herein can befrom about 10 mL to about 100 mL. For example, the starting volume ofthe biological sample injected in to the systems described herein can befrom about 10-90 mL, 10-80 mL, 10-70 mL, 10-60 mL, 10-50 mL, 10-40 mL,10-30 mL, 10-20 mL, 20-100 mL, 20-90 mL, 20-80 mL, 20-70 mL, 20-60 mL,20-50 mL, 20-40 mL, 20-30 mL, 30-100 mL, 30-90 mL, 30-80 mL, 30-70 mL,30-60 mL, 30-50 mL, 30-40 mL, 40-100 mL, 40-90 mL, 40-80 mL, 40-70 mL,40-60 mL, 40-50 mL, 50-100 mL, 50-90 mL, 50-80 mL, 50-70 mL, 50-60 mL,60-100 mL, 60-90 mL, 60-80 mL, 60-70 mL, 70-100 mL, 70-90 mL, 70-80 mL,80-100 mL, 80-90 mL, or 90-100 mL. In some embodiments, this process ora similar process can be performed in approximately 1 to 5 minutes, 2 to5 minutes, 3 to 6 minutes, 4 to 5 minutes, 5 to 10 minutes, 6 to 10minutes, 7 to 10 minutes, 8 to 10 minutes, 9 to 10 minutes, 10 to 15minutes, 15 to 20 minutes, 10 to 30 minutes, 15 to 35 minutes, 20 to 40minutes, or 30 to 45 minutes. The waste volume, containing low molecularweight proteins, including potentially pro-inflammatory cytokines andproteases, can be discarded. The resulting autologous plateletconcentrate can be drawn into a syringe and provided for mixing asneeded for clinical administration, such as to a chronic wound.

Channels and Modules

The channels and modules and housings described herein can be made ofany suitable material. Suitable materials include both liquid permeableand non-permeable materials, such as polyolefins, polyamides,polyesters, silicones, polyurethanes, epoxies, acrylics, polyacrylates,polysulfones, polymethacrylates, PEEK, polyimide and fluoropolymers suchas PTFE Teflon®, FEP Teflon®, Tefzel®, poly(vinylidene fluoride), PVDFand perfluoroalkoxy resins. Glass products including silica glass can beused. Ceramic can be used according to embodiments of the invention.Cellulosic products such as paper and reinforced paper containers canalso be used to form channels and modules according to the invention.

The size of the various modules and channels can be suitable for holdingthe volumes of samples described above. For example, the channels andmodules can be from about 2-2000 cm, 2-1000 cm, 2-500 cm, 2-100 cm, 2-50cm, 2-25 cm, 2-10 cm, 2-5 cm, 2-4 cm, 2-3 cm in length. For example, thechannels and modules can be from about 0.1-200 cm, 0.1-100 cm, 0.1-50cm, 0.1-10 cm, 0.1-5 cm, 0.1-2.5 cm, 0.1-1 cm, 0.1-0.5 cm, 0.1-0.4 cm,0.1-0.3 cm, or 0.1-0.2 cm in width. For example, the channels andmodules can have a diameter of from about 0.1-200 cm, 0.1-100 cm, 0.1-50cm, 0.1-10 cm, 0.1-5 cm, 0.1-2.5 cm, 0.1-1 cm, 0.1-0.5 cm, 0.1-0.4 cm,0.1-0.3 cm, or 0.1-0.2 cm.

Some embodiments of the invention further comprise a pump adapted to befluidly coupled with the filtration module either upstream of the inletof the filtration module or downstream of the outlet of the filtrationmodule. The pump is further adapted to produce a flow of the fluidsample that passes through the one or more filters of the filter module.In some embodiments of the invention, the filter module furthercomprises at least one reservoir

A particular embodiment of the filtration module is well suited toreceive a supernatant 2409 of a fluid sample such as blood (not shown)that has been centrifuged (FIG. 34). The filter module 2401 has a firstfilter 2410 coupled to the filter module inlet 2402. The supernatant is2409 received and pumped from a receiving reservoir 2405 into the firstfilter. The first filter 2410 is typically a cross-flow filterconfigured to retain molecules larger than about 500 kDa in a retentatereservoir 2406. The retentate reservoir 2406 may also be the same as thereceiving reservoir 2405 for receiving the supernatant 2409 of the fluidsample. The permeate 2420 of the first filter 2410 containing moleculessmaller than about 500 kDa is directed to a permeate reservoir 2430which may be a waste bag 2431. A2M is concentrated in this retentatereservoir 2406. A filter module outlet 2450 maybe coupled to theretentate reservoir 2406 such that the concentrated A2M 2460 may beextracted from the retentate reservoir 2406 or pumped back through thefirst filter 2410.

An embodiment of the system for concentrating A2M is shown in FIGS.35-37. A blood bag 2801 is shown containing the fluid sample 2802, whichtypically comprises blood. (FIG. 37). The fluid sample 2802 is extractedvia syringe(s) 2803 and centrifuged with centrifuge 2804. The resultantsupernatant 2805 containing A2M and other small molecules but not cellsor large molecules is then directed to the filtration module 3209, wherein the A2M is concentrated in to a serum 2810 or a plasma 2811 with atleast one filter 2808 selected to retain molecules of larger in sizethan about 50, 100, and/or 500 kDa. The filtration module 3209 may besimilar to the examples described herein.

An embodiment of the system for concentrating A2M is shown in (FIG. 39).A blood bag 3201 is shown containing the fluid sample 3202. The fluidsample 3202 is pumped via pump 3203 to the first filter 3210 of thefiltration module 3204. The first filter 3210 shown here is a cross-flowfilter configured to screen out cells, particles, and other moleculeslarger than 1 micron. The permeate 3212 of the first filter comprisingcomponents of the fluid sample smaller than 1 micron is directed to afirst permeate reservoir 3215. The retentate 3216 of the first filter isdirected to a first retentate reservoir 3216, in this particularembodiment the first retentate reservoir is also blood bag 3201. Thepermeate 3212 of the first filter is then directed via pump 3203 to thesecond filter 3220. The second filter is typically a cross-flow filterconfigured to retain molecules of weight more than about 500 kDa.Molecules of weight more than about 50, 100, and/or 500 kDa are retainedas a second retentate 3223 in a second retentate reservoir 3225. Thesecond retentate reservoir 3225 may be the same as the first permeatereservoir 3215. Permeate of the second filter 3224 is typically directedto a second permeate reservoir 3226 in some embodiments the secondpermeate reservoir 3226 is a waste bag 3230. The retentate of the secondfilter 3223 comprises the concentrated A2M. The pump 3203 may be fluidlyconnected to the filtration module up stream of the filtration moduleinlet 3206 or down-stream of the filtration module outlet 3207 orin-between inlet the filtration module 3206 and the filtration moduleoutlet 3207 or any combination thereof. The second retentate reservoir3225 has an access port 3229 for directing flow of the concentrated A2M(which is also the retentate of the second filter) back to the pump.Such port may be used to access the concentrated A2M, or the permeate ofthe first filter, or the retentate of the second filter for subsequentprocessing or harvesting of the concentrated A2M.

Methods of A2M Enrichment and Preparation of Autologous Compositions

Methods of enrichment of A2M from a subject are provided herein. Themethods can be used to produce any of the autologous compositions. Thesystems can be used with any of the methods described herein.

A method for enrichment of A2M from a biological sample, such as amammalian biological sample, can comprise flowing or passing abiological sample through one or more filters or membranes, such as oneor more hollow fiber filters and/or membranes with a particular poresize. Flowing or passing a sample through one or more filters cancomprise flowing the sample through 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore filters. A sample can be separated into one or more filtrates andone or more retentates, for example a first filtrate and a firstretentate. For example, a sample can be separated into 2, 3, 4, 5, 6, 7,8, 9, 10, or more filtrates and 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreretentates upon flowing or passing the sample through one or morefilters. For example, a sample can be separated into 2 or more filtratesand 2 or more retentates upon flowing or passing the sample through twoor more filters. An A2M enriched sample can be a first, second, third,fourth, fifth, or more retentate upon flowing or passing a biologicalsample through one or more filters. One or more A2M enriched samples orretentates can be diluted, such as with a diluent. A diluent can be aliquid or a solution, such as a hypotonic, hypertonic, or isotonicsolution. For example, a diluent can be a WFI solution or a salinesolution.

A method for enrichment of A2M from a biological sample, such as amammalian biological sample can comprise separating cells from acellular biological sample, such as blood. In some embodiments, redblood cells can be separated from white blood cells and platelets byperforming one or more centrifugation steps or by flowing a samplethrough a filter. White blood cells can be separated from platelets byperforming one or more centrifugation steps. One or more centrifugationcycles can be used or applied to provide centrifugal force to flow orpush a biological sample through one or more filters. Gravitational,centrifugal or mechanical force can also be used or applied to provideforce to flow or push a biological by flowing a sample through one ormore filters. Mechanical force can be a pump, centrifugal force, gaspressure, or any other force that is operable to provide enough force toflow a sample through one or more filters as described herein. Asupernatant of a centrifuged or filtered blood sample can contain A2M.In some embodiments, the supernatant can contain A2M and platelets. Insome embodiments, the supernatant can contain A2M and not containplatelets.

It is an object of the current invention to concentrate platelets and,in some embodiments, allow for the retention of platelet-released growthfactors, while, in some embodiments, using a molecular weight cutoff ofa filter and a tangential flow ultrafiltration (TFF) step to avoid theconcentration of potentially proinflammatory cytokines and catabolicproteases. After obtaining PRP, tangential flow ultrafiltration (TFF)can be used to concentrate platelets to a desired concentration rangeusing the methods described herein, resulting in an autologous plateletintegrated concentrate in which low molecular weight proteins, such ascytokines and proteases, such as those less than about 50, 100, and/or500 kDa in mass, have not been substantially concentrated. In someembodiments, filter parameters can be chosen to concentrate plateletsand to avoid concentration of cytokines, proteases, and potentiallyundesirable plasma proteins.

In some embodiments, it is preferable to separate red blood cells andwhite blood cells from the biological sample and retain platelets withinthe biological sample prior to flowing the sample through one or morefilters by performing one or more centrifugation steps. In someembodiments, it is preferable to separate red blood cells, white bloodcells, and platelets from the biological sample prior to flowing orpassing the sample through the one or more filter by performing one ormore centrifugation steps.

In some embodiments, it is preferable to separate red blood cells andwhite blood cells from the biological sample and retain platelets withinthe biological sample by flowing or passing the biological samplethrough one or more filters or membranes. In some embodiments, it ispreferable to separate red blood cells, white blood cells, and plateletsfrom the biological sample by flowing or passing the biological samplethrough one or more filters. The one or more filters used to remove thecells and other large particles from the biological sample can have apore size of at least about 0.1 μm. For example, the one or more filtersused to remove the cells and other large particles from the biologicalsample can have a pore size of at least about 0.2 μm, 0.3 μm, 0.4 μm,0.5 μm, 0.6 μm, 0.7 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm,1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm,2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, or 3 μm,or higher. As a non-limiting example, a biological sample, such asblood, can be flowed through one or more filters with a pore size of atleast about 0.2 μm wherein the red blood cells and white blood cells areretained by the filter and are in the retentate and non-cellularcomponents and platelets are not retained by the filter and are in thefiltrate. As another non-limiting example, a biological sample, such asblood, can be flowed through one or more filters with a pore size of atleast about 0.2 μm wherein the red blood cells, white blood cells, andplatelets are retained by the filter in the retentate and non-cellularcomponents are not retained by the filter and are in the filtrate.

In some embodiments, one or more of the filters can have a charge,immobilized molecules, or a combination thereof and can thereby enhancethe selectivity of the filters. Immobilized molecules can be antibodies,proteins, receptors, ligands, carbohydrates, nucleotides, RNA, DNA orany combination thereof. For example, enhancing the selectivity offilters can enhance the ability of a filter to retain A2M in theretentate upon flowing or passing a biological sample through the filteror, as another example, enhance the ability of a filter to not retainmolecules that are not A2M upon flowing a biological sample through thefilter.

Additionally, one or more filters with a molecular weight cut-off can beused and can allow a percentage of particles in the biological sample,such as cells and proteins, with a molecular weight higher than themolecular weight cut-off of the filter to be retained by the filter. Theretained sample can be a retentate and the sample that flows through thefilter can be a filtrate. A filter with a molecular weight cut-off canallow more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of particles in abiological sample with a molecular weight higher than the molecularweight cut-off of the filter to be retained by the filter. For example,one or more filters can retain about 100% of cells, cellular debris, ora combination thereof, from a blood sample and can retain less thanabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the proteins with a molecular weightless than about 50, 100, and/or 500 kDa from a biological samplecontaining A2M.

One or more filtrates can be passed through one or more other filters byapplying a gravitational, centrifugal, or mechanical force to the one ormore filtrates. A sample can be separated into one or more otherfiltrates and one or more other retentates, for example a secondfiltrate and a second retentate. For example, a sample can be separatedinto 2, 3, 4, 5, 6, 7, 8, 9, 10, or more other filtrates and 2, 3, 4, 5,6, 7, 8, 9, 10, or more other retentates upon flowing the sample through1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more other filters. For example, asample can be separated into 2 other filtrates and 2 other retentatesupon flowing or passing the sample through one or more other filters. AnA2M enriched sample can be a first, second, third, fourth, fifth, ormore other retentate upon flowing a biological sample through one ormore other filters. One or more other retentates, filtrates, or A2Menriched samples can be diluted, such as with a diluent. For example, adiluent can be a liquid, such as a WFI solution or a saline solution.

In some embodiments, after separating the cells from a biological samplethe resulting composition lacking red blood cells and white blood cells,and either lacking or containing a same, reduced or elevated plateletlevel, can be flowed or passed through one or more filters to obtain oneor more filtrates and retentates, such as an A2M enriched retentate orA2M concentrated retentate. The resulting one or more filtrates can beflowed through one or more other filters with a molecular weight cut-offof at most about 50, 100, and/or 500 kDa to obtain one or more otherfiltrates and retentates, such as an A2M enriched or concentratedretentate. For example, a filtrate lacking red blood cells and whiteblood cells, and either lacking or containing a same, reduced orelevated platelet level, can be flowed through one or more filters witha molecular weight cut-off of at most about 50 kDa, 55 kDa, 60 kDa, 65kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 110 kDa,120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa,200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa,280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa,360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa,440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa or lower to obtainone or more filtrates and retentates, such as an A2M enriched retentateor A2M concentrated retentate.

In some embodiments, a filtrate can comprise less than about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of the red blood cells in a biological sample. In someembodiments, a filtrate can comprise less than about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of the white blood cells in a biological sample. In someembodiments, a filtrate can comprise less than about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of the red and white blood cells in a biological sample. In someembodiments, a filtrate can comprise less than about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of the platelets in a biological sample. In some embodiments, afiltrate can comprise more than about or about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the platelets in a biological sample.

In some embodiments, a retentate can comprise more than about or about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% of the red blood cells in a biologicalsample. In some embodiments, a retentate can comprise more than about orabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the white blood cells in abiological sample. In some embodiments, a retentate can comprise morethan about or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the red and whiteblood cells in a biological sample. In some embodiments, a retentate cancomprise less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the platelets ina biological sample. In some embodiments, a retentate can comprise morethan about or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the platelets in abiological sample.

A retentate can be or comprise any of the compositions described, suchas an autologous A2M composition. For example, a retentate can comprisean elevated concentration of A2M compared to the concentration of A2Mfound in a biological sample. For example, a retentate can comprise anelevated concentration of A2M compared to the concentration of A2M foundin a biological sample and an elevated concentration of one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa foundin a biological sample. For example, a retentate can comprise a reducedconcentration of a protein with a molecular weight greater than about50, 100, and/or 500 kDa compared to the concentration of the proteinfound in a biological sample and an elevated concentration of one ormore proteins with molecular weight higher than 50, 100, and/or 500 kDafound in a biological sample. For example, a retentate can comprise anelevated concentration of A2M compared to the concentration of A2M foundin a biological sample and a reduced concentration of a protein with amolecular weight greater than about 50, 100, and/or 500 kDa compared tothe concentration of the protein found in a biological sample.

In some embodiments, a retentate can comprise less than about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of the red blood cells in a biological sample. In someembodiments, a retentate can comprise less than about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% of the white blood cells in a biological sample. In someembodiments, a retentate can comprise less than about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% of the red and white blood cells in a biological sample. Insome embodiments, a retentate can comprise less than about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of the platelets in a biological sample. In someembodiments, a retentate can comprise more than about or about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of the platelets in a biological sample.

For example, a concentration of A2M in a retentate obtained by flowing abiological sample lacking red blood cells, white blood cells andplatelets through one or more filters with a molecular weight cut-off ofat most about 50, 100, and/or 500 kDa can be at least about 1.1 timeshigher than the concentration of A2M found in a biological sample andthe concentration of one or more proteins with molecular weight higherthan 50, 100, and/or 500 kDa can be at least about 1.1 times higher thanthe concentration of the one or more proteins with molecular weighthigher than 50, 100, and/or 500 kDa found in a biological sample. Forexample, the concentration of A2M in a retentate obtained by flowing abiological sample lacking red blood cells, white blood cells andplatelets through one or more filters with a molecular weight cut-off ofat most about 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85kDa, 90 kDa, 95 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470kDa, 480 kDa, 490 kDa, 500 kDa, or lower, can be at least about 1.6,1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa canbe at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higher than theconcentration of the one or more proteins with molecular weight higherthan about 50, 100, and/or 500 kDa found in a biological sample. Forexample, the concentration of A2M in a retentate obtained by flowing abiological sample lacking red blood cells, white blood cells andplatelets through one or more filters with a molecular weight cut-off ofat most about 50, 100, and/or 500 kDa can be at least about 2 timeshigher than the concentration of A2M found in a biological sample andthe concentration of one or more proteins with molecular weight higherthan 50, 100, and/or 500 kDa can be at least about 2 times higher thanthe concentration of the one or more proteins with molecular weighthigher than about 50, 100, and/or 500 kDa found in a biological sample.

As another example, a retentate, such as an A2M enriched or concentratedretentate obtained by flowing or passing a composition, such as afiltrate lacking red blood cells and white blood cells, but not lackingplatelets, through one or more filters with a molecular weight cut-offof at most about 50, 100, and/or 500 kDa, can comprise an elevatedconcentration of A2M compared to the concentration of A2M found in abiological sample and an elevated concentration of one or more proteinswith molecular weight higher than 50, 100, and/or 500 kDa found in abiological sample. The concentration of A2M in a retentate obtained byflowing a biological sample lacking red blood cells and white bloodcells, but not lacking platelets, through one or more filters with amolecular weight cut-off of at most about 50, 100, and/or 500 kDa can beat least about 1.1 times higher than the concentration of A2M found in abiological sample and the concentration of one or more proteins withmolecular weight higher than 50, 100, and/or 500 kDa can be at leastabout 1.1 times higher than the concentration of the one or moreproteins with molecular weight higher than 50, 100, and/or 500 kDa foundin a biological sample. For example, The concentration of A2M in aretentate obtained by flowing a biological sample lacking red bloodcells and white blood cells, but not lacking platelets, through one ormore filters with a molecular weight cut-off of at most about 50 kDa, 55kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500kDa, or lower, can be at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times higher than the concentration of A2M found in a biological sampleand the concentration of one or more proteins with molecular weighthigher than 500 kDa can be at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times higher than the concentration of the one or more proteins withmolecular weight higher than about 500 kDa found in a biological sample.For example, the concentration of A2M in a retentate obtained by flowinga biological sample lacking red blood cells, white blood cells, but notlacking platelets, through one or more filters with a molecular weightcut-off of at most about 500 kDa can be at least about 2 times higherthan the concentration of A2M found in a biological sample and theconcentration of one or more proteins with molecular weight higher than500 kDa can be at least about 2 times higher than the concentration ofthe one or more proteins with molecular weight higher than about 500 kDafound in a biological sample.

In some embodiments, after passing a sample, such as a biological sampleor one or more filtrates, through one or more filters, at least about10% of particles, and proteins with a molecular weight less than about50, 100, and/or 500 kDa can be removed from the sample. For example, atleast about 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells,particles, and small proteins with a molecular weight less than about50, 100, and/or 500 kDa can be removed from the sample. For example, atleast about 20% of particles and proteins with a molecular weight lessthan about 50, 100, and/or 500 kDa can be removed from the sample. Atleast about 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells,particles, and proteins with a molecular weight less than about 50 kDa,55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa,100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa,180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa,260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa,340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa,420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa,or less can be removed from the sample. For example, at least about 20%of particles and small proteins with a molecular weight less than 50,100, and/or 500 kDa, can be removed from the sample. An autologouscomposition described herein can be isolated after passing a samplethrough one or more filters.

One or more additional non-blood derived components can be added to theone or more filtrates or retentates. Non-blood derived components can beadded before, during, or after isolation. A non-blood derived componentcan be an anti-coagulant. For example, an anti-coagulant can be EDTA,tri-sodium citrate, water for injection (WFI), or saline.

One or more additional blood products or blood-derived components can beadded to the one or more filtrates or retentates. Blood products orblood-derived components can be added before, during, or afterisolation. Blood products or blood derived components can be cells,peptides, proteins, DNA, RNA, carbohydrates, or other small molecules.For example, blood products or blood-derived components can be red bloodcells, white blood cells, or platelets.

Platelets can be isolated from a biological sample according to anymethod known in the art, such as by centrifugation of a blood sample.Red blood cells and white blood cells can be sedimented bycentrifugation at relatively low centrifugal force, for example, lessthan 1000 g. Platelets can be isolated by centrifugation of the plateletcontaining plasma obtained from a first centrifugation. A plateletcontaining plasma can be centrifuged, for example, between about 3000 gto 5000 g, to sediment platelets. The above procedure can also beperformed in one or more centrifugation steps.

In some embodiments, one or more other filtrates or retentates, forexample a second filtrate or second retentate can be collected. One ormore additional non-blood derived components can be added to the one ormore other filtrates or other retentates. Non-blood derived componentscan be added before, during, or after isolation. A non-blood derivedcomponent can be an anti-coagulant. For example, an anti-coagulant canbe EDTA, tri-sodium citrate, water for injection (WFI), or saline.

One or more additional blood products or blood-derived components can beadded to the one or more other filtrates or other retentates. Bloodproducts or blood-derived components can be added before, during, orafter flowing or passing the biological sample through one or morefilters. Blood products or blood-derived components can be cells,peptides, proteins, DNA, RNA, carbohydrates, or other small molecules.For example, blood products or blood-derived components can be red bloodcells, white blood cells, or platelets.

In some embodiments, an autologous composition produced from use of asystem as described herein can have minimal foaming, and thus besuitable for injection into a subject. In some embodiments, acomposition can have substantially no foaming. In some embodiments, acomposition can have a volume wherein less than 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%,0.00001%, or less of the total volume of the composition is foam.Methods of measuring the amount of foam are known in the art. Examplesinclude measuring specific conductivity, volume changes after removingfoam, foam volumes, rate of foam degeneration over time, viscosity,optical absorption, specific surface, and other measurements. Forexample, in some embodiments, determining the amount of foam in acomposition can comprise measuring the viscosity of the composition. Insome embodiments, foaming measurements can be compared to foamingmeasurements taken from a reference sample. The reference sample cancomprise a known amount of foaming.

Variant A2M Polypeptides Compositions for Treatment of Chronic Wounds

A2M is a general inhibitor of metalloproteases and other proteases suchas ADAMTS 4 and ADAMTS 5. These proteases and others produced as aresult of a wound, such as a chronic wound can be responsible for slowhealing of the wound or persistence of the wound. Any of the recombinantcompositions described herein can be used for treatment of a subjectwith a condition, disease, or chronic wound according to any of themethods described herein.

A2M is able to inactivate an enormous variety of proteases (includingserine-, cysteine-, and aspartic-metalloproteases). A2M can function asan inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. A2Mcan function as an inhibitor of coagulation by inhibiting thrombin.Human A2M has in its structure a 38 amino acid “bait” region. The baitregion varies widely in the amino acid number (27-52 amino acids) andsequence between animal species. Proteases binding and cleaving of thebait region can become bound to A2M. The protease-A2M complex can berecognized by macrophage receptors and cleared from the organism'ssystem. A2M is able to inhibit all four classes of proteases by a unique‘trapping’ mechanism. When a protease cleaves the bait region, aconformational change can be induced in the protein which can trap theprotease. The entrapped enzyme can remain active against low molecularweight substrates (activity against high molecular weight substrates canbe greatly reduced). Following cleavage in the bait region a thioesterbond can be hydrolyzed and can mediate the covalent binding of theprotein to the protease.

In one aspect, provided herein is a composition that can be a variantA2M polypeptide. A variant A2M polypeptide can be a recombinant protein,or fragments thereof, and can be produced in a host cell and purifiedfor use in treatment of chronic wounds. A variant A2M composition can bemore efficient in inhibiting proteases, have longer half-life, have aslower clearance factor, or any combination thereof compared to awild-type A2M. A variant A2M can be a recombinant protein, or a fragmentthereof, and can be produced in a host cell and purified. For example, avariant A2M recombinant protein can be produced in a host comprisingbacteria, yeast, fungi, insect, or mammalian cells, or a cell freesystem.

Variant A2M polypeptides or fragments thereof, can also be variants orposttranslationally modified variants of A2M. A2M variant polypeptidescan have an integer number of amino acid alterations such that theiramino acid sequence shares at least about 60%, 70%, 80%, 85%, 90%, 95%,97%, 98%, 99%, 99.5% or 100% identity with an amino acid sequence of awild type A2M polypeptide. In some embodiments, A2M variant polypeptidescan have an amino acid sequence sharing at least about 60%, 70%, 80%,85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity with the amino acidsequence of a wild type A2M polypeptide.

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The Smith Waterman algorithm can also be used to determine percentidentity. Exemplary parameters for amino acid sequence comparisoninclude the following: 1) algorithm from Needleman and Wunsch (J. Mol.Biol., 48:443-453 (1970)); 2) BLOSSUM62 comparison matrix from Hentikoffand Hentikoff (Proc. Nat. Acad. Sci. USA., 89:10915-10919 (1992)) 3) gappenalty=12; and 4) gap length penalty=4. A program useful with theseparameters can be publicly available as the “gap” program (GeneticsComputer Group, Madison, Wis.). The aforementioned parameters are thedefault parameters for polypeptide comparisons (with no penalty for endgaps). Alternatively, polypeptide sequence identity can be calculatedusing the following equation: % identity−(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps

Variant A2M polypeptides, or fragments thereof, include but are notlimited to, those containing as a primary amino acid sequence all orpart of the amino acid sequence encoded by SEQ ID NOs: 5-66, andfragments of these proteins, including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a silent change. The variant A2Mpolypeptides can include all or part of the amino acid sequence encodedby SEQ ID NO: 3. The variant A2M polypeptides can be, for example, anynumber of between 4-20, 20-50, 50-100, 100-300, 300-600, 600-1000,1000-1450 consecutive amino acids containing the amino acids sequencesof SEQ. ID NOs. 5-66. The variant A2M polypeptide can be less than orequal to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400,500, 600, 700, 800, 900, 1000, and 1450 amino acids in length andcontain, as part of the sequence: SEQ ID NOs: 5-66. Variant A2Mpolypeptides includes polypeptide sequences having at least 95%, 94%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%,79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%,65%, 64%, 63%, 62%, 61%, or 60% sequence identity or similarity to anyvariant A2M polypeptide containing one of SEQ ID NOs: 5-66.

The variant A2M polypeptides provided herein also include proteinscharacterized by amino acid sequences similar to those of purifiedproteins but into which modification are naturally provided ordeliberately engineered. For example, modifications, in the variant A2Mpeptide or variant A2M DNA sequence, can be made by those skilled in theart using known techniques. Modifications of interest in the proteinsequences can include the alteration, substitution, replacement,insertion or deletion of a selected amino acid residue in the codingsequence. For example, one or more of the cysteine residues can bedeleted or replaced with another amino acid to alter the conformation ofthe molecule. Techniques for such alteration, substitution, replacement,insertion or deletion are well known to those skilled in the art (see,e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration,substitution, replacement, insertion or deletion retains the desiredactivity of the protein. Regions of the protein that are important forthe protein function can be determined by various methods known in theart including the alanine-scanning method which involves systematicsubstitution of single or multiple amino acids with alanine, followed bytesting the resulting alanine-containing variant for biologicalactivity. This type of analysis can be used to determine the importanceof the substituted amino acid(s) in biological activity.

The bait region of A2M is a segment that is susceptible to proteolyticcleavage, and which, upon cleavage, initiates a conformational change inthe A2M molecule resulting in the collapse of the structure around theprotease. For the exemplary A2M sequences set forth in SEQ ID NO: 3, thebait region corresponds to amino acids 690-728. For the exemplary A2Msequences set forth in SEQ ID NO: 1 and 2, the bait region correspondsto the nucleotides encoding amino acids 690-728.

A variant A2M polypeptide can comprise a bait region of a variant A2Mpolypeptide. For example, a bait region of a variant A2M polypeptide canbe a mutant bait region, fragment of a bait region, a bait region fromanother species, an isoform of a bait region, or a bait regioncontaining multiple copies of one or more bait regions described herein,or any combination thereof. A bait region of a variant A2M polypeptidecan include a plurality of protease recognition sites arranged in seriesand can be arranged in any order.

A bait region of a variant A2M polypeptide can have one or more proteaserecognition sites. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moreprotease recognition sites. Protease recognition sites or substrate baitregions can be consensus sequences for serine proteases, threonineproteases, cysteine proteases, aspartate proteases, metalloproteases,glutamic acid proteases, or any combination thereof compared to a wildtype A2M protein. A variant A2M polypeptide can be characterized by anenhanced specific inhibition of serine proteases, threonine proteases,cysteine proteases, aspartate proteases, metalloproteases, glutamic acidproteases, or any combination thereof. A variant A2M polypeptide can becharacterized by an enhanced nonspecific inhibition of serine proteases,threonine proteases, cysteine proteases, aspartate proteases,metalloproteases, glutamic acid proteases, or any combination thereofcompared to a wild type A2M protein.

A bait region of a variant A2M polypeptide can have one or more mutantbase regions. For example, a bait region of a variant A2M polypeptidecan have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more mutant baseregions. A bait region of a variant A2M polypeptide can have one or morebait region fragments. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or morebait region fragments. A fragment of a bait region of a variant A2Mpolypeptide can be a fragment of any of SEQ ID NOs: 5-66.

A bait region of a variant A2M polypeptide can have one or more mutantamino acids that are different than those amino acids in a wild-type A2Mpolypeptide. For example, a bait region of a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more mutant amino acidsthat are different than those amino acids in a wild-type A2Mpolypeptide. A bait region of a variant A2M polypeptide can have one ormore mutant amino acid regions that are different than those regions ina wild-type A2M polypeptide. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moremutant amino acid regions that are different than those regions in awild-type A2M polypeptide. A mutant bait region of a variant A2Mpolypeptide can replace or substitute a bait region in a wild-type A2Mpolypeptide. A mutant bait region of a variant A2M polypeptide can beany of SEQ ID NOs: 5-66.

The A2M variant polypeptides provided herein also include A2M variantproteins characterized by amino acid sequences similar to those ofpurified A2M variant. Isolated or purified variant A2M polypeptides canhave one or more amino acid residues within the polypeptide that aresubstituted by another amino acid of a similar polarity that acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence can be selected from other members ofthe class to which the amino acid belongs. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. The aromatic aminoacids include phenylalanine, tryptophan, and tyrosine.

A bait region of a variant A2M polypeptide can have one or more baitregion isoforms. For example, a bait region of a variant A2M polypeptidecan have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait regionisoforms. A bait region of a variant A2M polypeptide can have one ormore mutant or engineered bait regions. For example, a bait region of avariant A2M polypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or10 or more mutant or engineered bait regions.

A bait region of a variant A2M polypeptide can have one or more copiesof one or more bait regions. The one or more bait regions can be thesame bait regions (repeats), different bait regions, or any combinationthereof. For example, a bait region of a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more copies of one ormore bait regions, wherein the one or more bait regions can be the samebait regions (repeats), different bait regions, or any combinationthereof.

A variant A2M polypeptide can comprise one or more bait regions derivedfrom different organisms, different species of an organism, or acombination thereof. For example, a variant A2M polypeptide can have 2or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait regions derived fromdifferent organisms, different species of an organism, or a combinationthereof. One or more bait regions derived from different organisms canbe derived from one or more different organisms and not from differentspecies of an organism. For example, one or more modified bait regionscan be derived from 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moredifferent organisms and not contain 2 or more bait regions derived fromdifferent species of an organism. One or more bait regions derived fromdifferent species of an organism can be derived from one or moredifferent species of an organism and not from different organisms. Forexample, one or more modified bait regions can be derived from 2 ormore, or 3, 4, 5, 6, 7, 8, 9, or 10 or more different species of anorganism and not contain 2 or more bait regions derived from differentorganism. The modified bait regions can be derived from any animal,insect, plant, bacteria, viral, yeast, fish, reptile, amphibian, orfungi. The modified bait regions can be derived from any animal with A2Mor homologous protein, such as pig, mouse, rat, rabbit, cat, dog, frog,monkey, horse or goat.

A variant A2M polypeptide can comprise one or more bait regions ofvariant A2M polypeptides. For example, a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait region ofvariant A2M polypeptides. One or more bait region of a variant A2Mpolypeptides can be derived from one or more different species. Forexample, one or more bait regions of variant A2M polypeptides can bederived from 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more differentspecies. The bait region of variant A2M polypeptides can be derived fromany animal, insect, plant, bacteria, viral, yeast, fish, reptile,amphibian, or fungi species.

A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from oneor more proteins other than A2M.

A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from A2M.A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from oneor more non-natural protein sequences. The non-natural protein sequencescan comprise one or more protease recognition sites in series and canfunction as bait for proteases. A variant A2M polypeptide can have aplurality of protease recognition sites that can be one or more proteasesubstrate bait regions from or any of the combination of bait regionsdescribed herein. A variant A2M polypeptide can have any number ofprotease bait regions arranged in series. A variant A2M polypeptide canhave any number of protease bait regions from any species and can bearranged in series. One or more protease substrate bait regions from oneor more proteins other than A2M or from the one or more non-naturalprotein sequences can be a suicide inhibitor. For example, a variant A2Mpolypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore suicide inhibitor bait regions. A suicide inhibitor can be operableto covalently attach a protease to A2M.

A variant A2M polypeptide can be characterized by at least about a 10%increase in protease inhibitory effectiveness compared to the proteaseinhibitory effectiveness of a wild type A2M protein. For example, avariant A2M polypeptide can be characterized by at least about a 20,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% increase in protease inhibitory effectiveness when comparedto the protease inhibitory effectiveness of a wild type A2M protein. Avariant A2M polypeptide can be characterized by an increase in proteaseinhibitory effectiveness compared to the protease inhibitoryeffectiveness of a wild type A2M protein. For example, a variant A2Mpolypeptide can be characterized by an 1.2, 1.2, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times increase in protease inhibitoryeffectiveness compared to the protease inhibitory effectiveness of awild type A2M protein.

A variant A2M polypeptide can be characterized as having an increasedability to inhibit one or more proteases compared to a wild-type A2Mpolypeptide. A variant A2M polypeptide can have an ability to inhibitone or more proteases that is at least 1.5 times higher than the abilityof a wild-type A2M polypeptide to inhibit the one or more proteases. Forexample, a variant A2M polypeptide can have an ability to inhibit one ormore proteases that is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times higher than theability of a wild-type A2M polypeptide to inhibit the one or moreproteases. A variant A2M polypeptide can have an ability to inhibit oneor more proteases that is from 1.5-100 times higher than the ability ofa wild-type A2M polypeptide to inhibit the one or more proteases. Forexample, a variant A2M polypeptide can have an ability to inhibit one ormore proteases that is from 1.6-100, 1.7-100, 1.8-100, 1.9-100, 2-100,2.1-100, 2.2-100, 2.3-100, 2.4-100, 2.5-100, 2.6-100, 2.7-100, 2.8-100,2.9-100, 3.0-100, 3.1-100, 3.2-100, 3.3-100, 3.4-100, 3.5-100, 3.6-100,3.7-100, 3.8-100, 3.9-100, 4-100, 5-100, 6-100, 7-100, 8-100, 9-100,10-100, 11-100, 12-100, 13-100, 14-100, 15-100, 16-100, 17-100, 18-100,19-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 60-100,70-100, 80-100, 90-100, 1.5-90, 1.6-90, 1.7-90, 1.8-90, 1.9-90, 2-90,2.1-90, 2.2-90, 2.3-90, 2.4-90, 2.5-90, 2.6-90, 2.7-90, 2.8-90, 2.9-90,3.0-90, 3.1-90, 3.2-90, 3.3-90, 3.4-90, 3.5-90, 3.6-90, 3.7-90, 3.8-90,3.9-90, 4-90, 5-90, 6-90, 7-90, 8-90, 9-90, 10-90, 11-90, 12-90, 13-90,14-90, 15-90, 16-90, 17-90, 18-90, 19-90, 20-90, 25-90, 30-90, 35-90,40-90, 45-90, 50-90, 60-90, 70-90, 80-90, 1.5-80, 1.6-80, 1.7-80,1.8-80, 1.9-80, 2-80, 2.1-80, 2.2-80, 2.3-80, 2.4-80, 2.5-80, 2.6-80,2.7-80, 2.8-80, 2.9-80, 3.0-80, 3.1-80, 3.2-80, 3.3-80, 3.4-80, 3.5-80,3.6-80, 3.7-80, 3.8-80, 3.9-80, 4-80, 5-80, 6-80, 7-80, 8-80, 9-80,10-80, 11-80, 12-80, 13-80, 14-80, 15-80, 16-80, 17-80, 18-80, 19-80,20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 60-80, 70-80, 1.5-70,1.6-70, 1.7-70, 1.8-70, 1.9-70, 2-70, 2.1-70, 2.2-70, 2.3-70, 2.4-70,2.5-70, 2.6-70, 2.7-70, 2.8-70, 2.9-70, 3.0-70, 3.1-70, 3.2-70, 3.3-70,3.4-70, 3.5-70, 3.6-70, 3.7-70, 3.8-70, 3.9-70, 4-70, 5-70, 6-70, 7-70,8-70, 9-70, 10-70, 11-70, 12-70, 13-70, 14-70, 15-70, 16-70, 17-70,18-70, 19-70, 20-70, 25-70, 30-70, 35-70, 40-70, 45-70, 50-70, 60-70,1.5-60, 1.6-60, 1.7-60, 1.8-60, 1.9-60, 2-60, 2.1-60, 2.2-60, 2.3-60,2.4-60, 2.5-60, 2.6-60, 2.7-60, 2.8-60, 2.9-60, 3.0-60, 3.1-60, 3.2-60,3.3-60, 3.4-60, 3.5-60, 3.6-60, 3.7-60, 3.8-60, 3.9-60, 4-60, 5-60,6-60, 7-60, 8-60, 9-60, 10-60, 11-60, 12-60, 13-60, 14-60, 15-60, 16-60,17-60, 18-60, 19-60, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60,1.5-50, 1.6-50, 1.7-50, 1.8-50, 1.9-50, 2-50, 2.1-50, 2.2-50, 2.3-50,2.4-50, 2.5-50, 2.6-50, 2.7-50, 2.8-50, 2.9-50, 3.0-50, 3.1-50, 3.2-50,3.3-50, 3.4-50, 3.5-50, 3.6-50, 3.7-50, 3.8-50, 3.9-50, 4-50, 5-50,6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50, 13-50, 14-50, 15-50, 16-50,17-50, 18-50, 19-50, 20-50, 25-50, 30-50, 35-50, 40-50, 1.5-40, 1.6-40,1.7-40, 1.8-40, 1.9-40, 2-40, 2.1-40, 2.2-40, 2.3-40, 2.4-40, 2.5-40,2.6-40, 2.7-40, 2.8-40, 2.9-40, 3.0-40, 3.1-40, 3.2-40, 3.3-40, 3.4-40,3.5-40, 3.6-40, 3.7-40, 3.8-40, 3.9-40, 4-40, 5-40, 6-40, 7-40, 8-40,9-40, 10-40, 11-40, 12-40, 13-40, 14-40, 15-40, 16-40, 17-40, 18-40,19-40, 20-40, 25-40, 30-40, 1.5-30, 1.6-30, 1.7-30, 1.8-30, 1.9-30,2-30, 2.1-30, 2.2-30, 2.3-30, 2.4-30, 2.5-30, 2.6-30, 2.7-30, 2.8-30,2.9-30, 3.0-30, 3.1-30, 3.2-30, 3.3-30, 3.4-30, 3.5-30, 3.6-30, 3.7-30,3.8-30, 3.9-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30,13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 1.5-20, 1.6-20,1.7-20, 1.8-20, 1.9-20, 2-20, 2.1-20, 2.2-20, 2.3-20, 2.4-20, 2.5-20,2.6-20, 2.7-20, 2.8-20, 2.9-20, 3.0-20, 3.1-20, 3.2-20, 3.3-20, 3.4-20,3.5-20, 3.6-20, 3.7-20, 3.8-20, 3.9-20, 4-20, 5-20, 6-20, 7-20, 8-20,9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 1.5-15, 1.6-15, 1.7-15,1.8-15, 1.9-15, 2-15, 2.1-15, 2.2-15, 2.3-15, 2.4-15, 2.5-15, 2.6-15,2.7-15, 2.8-15, 2.9-15, 3.0-15, 3.1-15, 3.2-15, 3.3-15, 3.4-15, 3.5-15,3.6-15, 3.7-15, 3.8-15, 3.9-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15,10-15, 11-15, 12-15, 13-15, 14-15, 1.5-10, 1.6-10, 1.7-10, 1.8-10,1.9-10, 2-10, 2.1-10, 2.2-10, 2.3-10, 2.4-10, 2.5-10, 2.6-10, 2.7-10,2.8-10, 2.9-10, 3.0-10, 3.1-10, 3.2-10, 3.3-10, 3.4-10, 3.5-10, 3.6-10,3.7-10, 3.8-10, 3.9-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1.5-9,1.6-9, 1.7-9, 1.8-9, 1.9-9, 2-9, 2.1-9, 2.2-9, 2.3-9, 2.4-9, 2.5-9,2.6-9, 2.7-9, 2.8-9, 2.9-9, 3.0-9, 3.1-9, 3.2-9, 3.3-9, 3.4-9, 3.5-9,3.6-9, 3.7-9, 3.8-9, 3.9-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1.5-8, 1.6-8,1.7-8, 1.8-8, 1.9-8, 2-8, 2.1-8, 2.2-8, 2.3-8, 2.4-8, 2.5-8, 2.6-8,2.7-8, 2.8-8, 2.9-8, 3.0-8, 3.1-8, 3.2-8, 3.3-8, 3.4-8, 3.5-8, 3.6-8,3.7-8, 3.8-8, 3.9-8, 4-8, 5-8, 6-8, 7-8, 1.5-7, 1.6-7, 1.7-7, 1.8-7,1.9-7, 2-7, 2.1-7, 2.2-7, 2.3-7, 2.4-7, 2.5-7, 2.6-7, 2.7-7, 2.8-7,2.9-7, 3.0-7, 3.1-7, 3.2-7, 3.3-7, 3.4-7, 3.5-7, 3.6-7, 3.7-7, 3.8-7,3.9-7, 4-7, 5-7, 6-7, 1.5-6, 1.6-6, 1.7-6, 1.8-6, 1.9-6, 2-6, 2.1-6,2.2-6, 2.3-6, 2.4-6, 2.5-6, 2.6-6, 2.7-6, 2.8-6, 2.9-6, 3.0-6, 3.1-6,3.2-6, 3.3-6, 3.4-6, 3.5-6, 3.6-6, 3.7-6, 3.8-6, 3.9-6, 4-6, 5-6, 1.5-5,1.6-5, 1.7-5, 1.8-5, 1.9-5, 2-5, 2.1-5, 2.2-5, 2.3-5, 2.4-5, 2.5-5,2.6-5, 2.7-5, 2.8-5, 2.9-5, 3.0-5, 3.1-5, 3.2-5, 3.3-5, 3.4-5, 3.5-5,3.6-5, 3.7-5, 3.8-5, 3.9-5, 4-5, 1.5-4, 1.6-4, 1.7-4, 1.8-4, 1.9-4, 2-4,2.1-4, 2.2-4, 2.3-4, 2.4-4, 2.5-4, 2.6-4, 2.7-4, 2.8-4, 2.9-4, 3.0-4,3.1-4, 3.2-4, 3.3-4, 3.4-4, 3.5-4, 3.6-4, 3.7-4, 3.8-4, 3.9-4, 1.5-3,1.6-3, 1.7-3, 1.8-3, 1.9-3, 2-3, 2.1-3, 2.2-3, 2.3-3, 2.4-3, 2.5-3,2.6-3, 2.7-3, 2.8-3, 2.9-3, 1.5-2, 1.6-2, 1.7-2, 1.8-2, or 1.9-2 timeshigher than the ability of a wild-type A2M polypeptide to inhibit theone or more proteases.

The one or more proteases can include a matrix metalloprotease, such asMMP1 (Interstitial collagenase), MMP2 (Gelatinase-A), MMP3 (Stromelysin1), MMP7 (Matrilysin, PUMP 1), MMP8 (Neutrophil collagenase), MMP9(Gelatinase-B), MMP10 (Stromelysin 2), MMP11), Stromelysin 3), MMP12(Macrophage metalloelastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP),MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18 (Collagenase 4,xcol4, xenopus collagenase), MMP19 (RASI-1, stromelysin-4), MMP20(Enamelysin), MMP21 (X-MMP), MMP23A (CA-MMP), MMP23B MMP24 (MT5-MMP),MMP25 (MT6-MMP), MMP26 (Matrilysin-2, endometase), MMP27 (MMP-22,C-MMP), MMP28 (Epilysin); A Disintegrin and Metalloproteinase withThrombospondin Motifs protease, such as ADAMTS1, ADAMTS2, ADAMTS3,ADAMTS4, ADAMTS5 (ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8 (METH-2),ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16,ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20; chymotrypsin; trypsin; elastase;compliment factors; clotting factors; thrombin; plasmin; subtilisin;Neprilysin; Procollagen peptidase; Thermolysin; Pregnancy-associatedplasma protein A; Bone morphogenetic protein 1; Lysostaphin; Insulindegrading enzyme; ZMPSTE2; and acetylcholinesterase.

A variant A2M polypeptide can be characterized as having an increasedability to prevent FAC formation compared to a wild-type A2Mpolypeptide. A variant A2M polypeptide can have an ability to preventFAC formation that is at least 1.5 times higher than the ability of awild-type A2M polypeptide to prevent FAC formation. For example, avariant A2M polypeptide can have an ability to prevent FAC formationthat is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, or 100 times higher than the ability of awild-type A2M polypeptide to prevent FAC formation. A variant A2Mpolypeptide can have an ability to prevent FAC formation that is from1.5-100 times higher than the ability of a wild-type A2M polypeptide toprevent FAC formation. For example, a variant A2M polypeptide can havean ability to prevent FAC formation that is from 1.6-100, 1.7-100,1.8-100, 1.9-100, 2-100, 2.1-100, 2.2-100, 2.3-100, 2.4-100, 2.5-100,2.6-100, 2.7-100, 2.8-100, 2.9-100, 3.0-100, 3.1-100, 3.2-100, 3.3-100,3.4-100, 3.5-100, 3.6-100, 3.7-100, 3.8-100, 3.9-100, 4-100, 5-100,6-100, 7-100, 8-100, 9-100, 10-100, 11-100, 12-100, 13-100, 14-100,15-100, 16-100, 17-100, 18-100, 19-100, 20-100, 25-100, 30-100, 35-100,40-100, 45-100, 50-100, 60-100, 70-100, 80-100, 90-100, 1.5-90, 1.6-90,1.7-90, 1.8-90, 1.9-90, 2-90, 2.1-90, 2.2-90, 2.3-90, 2.4-90, 2.5-90,2.6-90, 2.7-90, 2.8-90, 2.9-90, 3.0-90, 3.1-90, 3.2-90, 3.3-90, 3.4-90,3.5-90, 3.6-90, 3.7-90, 3.8-90, 3.9-90, 4-90, 5-90, 6-90, 7-90, 8-90,9-90, 10-90, 11-90, 12-90, 13-90, 14-90, 15-90, 16-90, 17-90, 18-90,19-90, 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 60-90, 70-90,80-90, 1.5-80, 1.6-80, 1.7-80, 1.8-80, 1.9-80, 2-80, 2.1-80, 2.2-80,2.3-80, 2.4-80, 2.5-80, 2.6-80, 2.7-80, 2.8-80, 2.9-80, 3.0-80, 3.1-80,3.2-80, 3.3-80, 3.4-80, 3.5-80, 3.6-80, 3.7-80, 3.8-80, 3.9-80, 4-80,5-80, 6-80, 7-80, 8-80, 9-80, 10-80, 11-80, 12-80, 13-80, 14-80, 15-80,16-80, 17-80, 18-80, 19-80, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80,50-80, 60-80, 70-80, 1.5-70, 1.6-70, 1.7-70, 1.8-70, 1.9-70, 2-70,2.1-70, 2.2-70, 2.3-70, 2.4-70, 2.5-70, 2.6-70, 2.7-70, 2.8-70, 2.9-70,3.0-70, 3.1-70, 3.2-70, 3.3-70, 3.4-70, 3.5-70, 3.6-70, 3.7-70, 3.8-70,3.9-70, 4-70, 5-70, 6-70, 7-70, 8-70, 9-70, 10-70, 11-70, 12-70, 13-70,14-70, 15-70, 16-70, 17-70, 18-70, 19-70, 20-70, 25-70, 30-70, 35-70,40-70, 45-70, 50-70, 60-70, 1.5-60, 1.6-60, 1.7-60, 1.8-60, 1.9-60,2-60, 2.1-60, 2.2-60, 2.3-60, 2.4-60, 2.5-60, 2.6-60, 2.7-60, 2.8-60,2.9-60, 3.0-60, 3.1-60, 3.2-60, 3.3-60, 3.4-60, 3.5-60, 3.6-60, 3.7-60,3.8-60, 3.9-60, 4-60, 5-60, 6-60, 7-60, 8-60, 9-60, 10-60, 11-60, 12-60,13-60, 14-60, 15-60, 16-60, 17-60, 18-60, 19-60, 20-60, 25-60, 30-60,35-60, 40-60, 45-60, 50-60, 1.5-50, 1.6-50, 1.7-50, 1.8-50, 1.9-50,2-50, 2.1-50, 2.2-50, 2.3-50, 2.4-50, 2.5-50, 2.6-50, 2.7-50, 2.8-50,2.9-50, 3.0-50, 3.1-50, 3.2-50, 3.3-50, 3.4-50, 3.5-50, 3.6-50, 3.7-50,3.8-50, 3.9-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50,13-50, 14-50, 15-50, 16-50, 17-50, 18-50, 19-50, 20-50, 25-50, 30-50,35-50, 40-50, 1.5-40, 1.6-40, 1.7-40, 1.8-40, 1.9-40, 2-40, 2.1-40,2.2-40, 2.3-40, 2.4-40, 2.5-40, 2.6-40, 2.7-40, 2.8-40, 2.9-40, 3.0-40,3.1-40, 3.2-40, 3.3-40, 3.4-40, 3.5-40, 3.6-40, 3.7-40, 3.8-40, 3.9-40,4-40, 5-40, 6-40, 7-40, 8-40, 9-40, 10-40, 11-40, 12-40, 13-40, 14-40,15-40, 16-40, 17-40, 18-40, 19-40, 20-40, 25-40, 30-40, 1.5-30, 1.6-30,1.7-30, 1.8-30, 1.9-30, 2-30, 2.1-30, 2.2-30, 2.3-30, 2.4-30, 2.5-30,2.6-30, 2.7-30, 2.8-30, 2.9-30, 3.0-30, 3.1-30, 3.2-30, 3.3-30, 3.4-30,3.5-30, 3.6-30, 3.7-30, 3.8-30, 3.9-30, 4-30, 5-30, 6-30, 7-30, 8-30,9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30,19-30, 20-30, 1.5-20, 1.6-20, 1.7-20, 1.8-20, 1.9-20, 2-20, 2.1-20,2.2-20, 2.3-20, 2.4-20, 2.5-20, 2.6-20, 2.7-20, 2.8-20, 2.9-20, 3.0-20,3.1-20, 3.2-20, 3.3-20, 3.4-20, 3.5-20, 3.6-20, 3.7-20, 3.8-20, 3.9-20,4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20,15-20, 1.5-15, 1.6-15, 1.7-15, 1.8-15, 1.9-15, 2-15, 2.1-15, 2.2-15,2.3-15, 2.4-15, 2.5-15, 2.6-15, 2.7-15, 2.8-15, 2.9-15, 3.0-15, 3.1-15,3.2-15, 3.3-15, 3.4-15, 3.5-15, 3.6-15, 3.7-15, 3.8-15, 3.9-15, 4-15,5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15, 14-15, 1.5-10,1.6-10, 1.7-10, 1.8-10, 1.9-10, 2-10, 2.1-10, 2.2-10, 2.3-10, 2.4-10,2.5-10, 2.6-10, 2.7-10, 2.8-10, 2.9-10, 3.0-10, 3.1-10, 3.2-10, 3.3-10,3.4-10, 3.5-10, 3.6-10, 3.7-10, 3.8-10, 3.9-10, 4-10, 5-10, 6-10, 7-10,8-10, 9-10, 1.5-9, 1.6-9, 1.7-9, 1.8-9, 1.9-9, 2-9, 2.1-9, 2.2-9, 2.3-9,2.4-9, 2.5-9, 2.6-9, 2.7-9, 2.8-9, 2.9-9, 3.0-9, 3.1-9, 3.2-9, 3.3-9,3.4-9, 3.5-9, 3.6-9, 3.7-9, 3.8-9, 3.9-9, 4-9, 5-9, 6-9, 7-9, 8-9,1.5-8, 1.6-8, 1.7-8, 1.8-8, 1.9-8, 2-8, 2.1-8, 2.2-8, 2.3-8, 2.4-8,2.5-8, 2.6-8, 2.7-8, 2.8-8, 2.9-8, 3.0-8, 3.1-8, 3.2-8, 3.3-8, 3.4-8,3.5-8, 3.6-8, 3.7-8, 3.8-8, 3.9-8, 4-8, 5-8, 6-8, 7-8, 1.5-7, 1.6-7,1.7-7, 1.8-7, 1.9-7, 2-7, 2.1-7, 2.2-7, 2.3-7, 2.4-7, 2.5-7, 2.6-7,2.7-7, 2.8-7, 2.9-7, 3.0-7, 3.1-7, 3.2-7, 3.3-7, 3.4-7, 3.5-7, 3.6-7,3.7-7, 3.8-7, 3.9-7, 4-7, 5-7, 6-7, 1.5-6, 1.6-6, 1.7-6, 1.8-6, 1.9-6,2-6, 2.1-6, 2.2-6, 2.3-6, 2.4-6, 2.5-6, 2.6-6, 2.7-6, 2.8-6, 2.9-6,3.0-6, 3.1-6, 3.2-6, 3.3-6, 3.4-6, 3.5-6, 3.6-6, 3.7-6, 3.8-6, 3.9-6,4-6, 5-6, 1.5-5, 1.6-5, 1.7-5, 1.8-5, 1.9-5, 2-5, 2.1-5, 2.2-5, 2.3-5,2.4-5, 2.5-5, 2.6-5, 2.7-5, 2.8-5, 2.9-5, 3.0-5, 3.1-5, 3.2-5, 3.3-5,3.4-5, 3.5-5, 3.6-5, 3.7-5, 3.8-5, 3.9-5, 4-5, 1.5-4, 1.6-4, 1.7-4,1.8-4, 1.9-4, 2-4, 2.1-4, 2.2-4, 2.3-4, 2.4-4, 2.5-4, 2.6-4, 2.7-4,2.8-4, 2.9-4, 3.0-4, 3.1-4, 3.2-4, 3.3-4, 3.4-4, 3.5-4, 3.6-4, 3.7-4,3.8-4, 3.9-4, 1.5-3, 1.6-3, 1.7-3, 1.8-3, 1.9-3, 2-3, 2.1-3, 2.2-3,2.3-3, 2.4-3, 2.5-3, 2.6-3, 2.7-3, 2.8-3, 2.9-3, 1.5-2, 1.6-2, 1.7-2,1.8-2, or 1.9-2 times higher than the ability of a wild-type A2Mpolypeptide to prevent FAC formation.

One aspect of the invention is a method for determining the enhancedinhibition of a protease by a variant A2M polypeptide comprising: a)providing a variant A2M polypeptide comprising a sequence of one or moreof SEQ ID NOs 5-66; b) contacting the variant A2M polypeptide with theprotease and a substrate cleaved by the protease; c) contacting awild-type A2M polypeptide with the protease and the substrate cleaved bythe protease; and d) comparing the amount of cleavage of the substratefrom step b) to the amount of cleavage of the substrate from step c),thereby determining the enhanced inhibition of the protease by thevariant A2M polypeptide.

Enzymatic glycoconjugation reactions can be targeted to glycosylationsites and to residues that are attached to glycosylation sites. Thetargeted glycosylation sites can be sites native to a wild-type A2Mprotein, native to a variant A2M polypeptide or, alternatively, they canbe introduced into a wild-type A2M or variant A2M polypeptide bymutation. Thus, a method for increasing the in vivo half-life of avariant A2M polypeptide is provided by the methods of the invention.

A variant A2M polypeptide can include an amino acid sequence thatmutated to insert, remove or relocate one or more glycosylation site inthe protein. When a site is added or relocated, it is not present or notpresent in a selected location in the wild-type A2M peptide. The mutantglycosylation site can be a point of attachment for a modified glycosylresidue that can be enzymatically conjugated to the glycosylation site.Using the methods of the invention, the glycosylation site can beshifted to any efficacious position on the peptide. For example, if thenative glycosylation site is sufficiently proximate or within the baitregion of variant A2M polypeptide peptide that conjugation interfereswith the ability to bind a protease, inhibit a protease, or acombination thereof, it is within the scope of the invention to engineera variant A2M polypeptide that includes a glycosylation site as modifiedor removed from the bait as necessary to provide a biologically activevariant A2M polypeptide.

Any glycosyltransferase or method of their use known in the art can beused for in vitro enzymatic synthesis of variant A2M polypeptides withcustom designed glycosylation patterns, various glycosyl structures, ora combination thereof possible. See, for example, U.S. Pat. Nos.5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826;US2003180835; and WO 03/031464.

The present invention provides methods of improving or lengthening thein vivo half-lives of variant A2M polypeptides by conjugating awater-soluble polymer to the variant A2M polypeptides through an intactglycosyl linking group. In an exemplary embodiment, covalent attachmentof polymers, such as polyethylene glycol (PEG), to such variant A2Mpolypeptides affords variant A2M polypeptides having in vivo residencetimes, and pharmacokinetic and pharmacodynamic properties, enhancedrelative to the unconjugated variant A2M polypeptide.

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein. The polymer backbone can be linear or branched. Branchedpolymer backbones are generally known in the art. Typically, a branchedpolymer has a central branch core moiety and a plurality of linearpolymer chains linked to the central branch core. PEG is commonly usedin branched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(n) in which R represents the core moiety,such as glycerol or pentaerythritol, and n represents the number ofarms. Many other polymers are also suitable for the invention. Examplesof suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers of ethylene glycol and propylene glycol and the like,poly(oxyethylated polyol), poly(olefinic alcohol),polyvinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine) and copolymers, terpolymers, and mixturesthereof. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 100 Da toabout 100,000 Da often from about 6,000 Da to about 80,000 Da.

A variant A2M polypeptide can further comprise PEG. A variant A2Mpolypeptide can have one or more mutant or modified glycosylation sites.The modified glycosylation sites can comprise PEG. For example, avariant A2M polypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or more mutant or modified glycosylation sites. The conjugationor addition of PEG to a variant A2M polypeptide with one or moremodified or abnormal glycosylation sites can result in a variant A2Mpolypeptide with a longer half-life than the half-life of a wild-typeA2M protein without PEG when disposed within or on a wound of a subject,such as a chronic wound of a subject. The conjugation or addition of PEGto a variant A2M polypeptide with one or more modified or abnormalglycosylation sites can result in a variant A2M polypeptide with alonger half-life than the half-life of a variant A2M polypeptide withoutone or more modified glycosylation sites without PEG when disposed wwithin or on a wound of a subject, such as a chronic wound of a subject.The conjugation or addition of PEG to a variant A2M polypeptide with oneor more modified or abnormal glycosylation sites can result in a variantA2M polypeptide with a longer half-life than the half-life of a variantA2M polypeptide with one or more modified glycosylation sites withoutPEG when within or on a wound of a subject, such as a chronic wound of asubject. For example, a variant A2M polypeptide with one or moremodified or abnormal glycosylation sites with PEG can have half-lifethat is 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the half-life of awild type A2M protein without PEG, a variant A2M polypeptide with one ormore modified glycosylation sites without PEG, or a variant A2Mpolypeptide without one or more modified glycosylation sites withoutPEG. For example, a variant A2M polypeptide with one or more modified orabnormal glycosylation sites with PEG can have half-life that is 2 timesthe half-life of a wild type A2M protein composition with one or moremodified or abnormal glycosylation sites without PEG when disposedwithin or on a wound of a subject, such as a chronic wound of a subject.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” can be intended nucleotide fragments which differfrom a nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

Fragments of the A2M variants of the present invention which are capableof exhibiting biological activity are also encompassed by the presentinvention. Fragments of the A2M variants can be in linear form or theycan be cyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments can be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. The presentinvention also provides both full-length and mature forms (for example,without a signal sequence or precursor sequence) of the disclosed A2Mvariants. The protein coding sequence can be identified in the sequencelisting by translation of the disclosed nucleotide sequences. The matureform of such A2M variants can be obtained by expression of a full-lengthpolynucleotide in a suitable mammalian cell or other host cell. Thesequence of the mature form of the A2M variants can be also determinablefrom the amino acid sequence of the full-length form. Where A2M variantsof the present invention are membrane bound, soluble forms of the A2Mvariants are also provided. In such forms, part or all of the regionscausing the A2M variants to be membrane bound are deleted so that theA2M variants are fully secreted from the cell in which it can beexpressed. A2M variant compositions of the present invention can furthercomprise an acceptable carrier, such as a hydrophilic, e.g.,pharmaceutically acceptable, carrier.

Variant A2M Polynucleotide Compositions

As used herein, “A2M polynucleotide,” when used with reference to SEQ IDNOs: 1 or 2, means the polynucleotide sequence of SEQ ID NO: 1 or 2, orfragments thereof, as well as any nucleic acid variants which includeone or more insertions, deletions, mutations, or a combination thereof.The insertions, deletions, and mutations are preferably within thepolynucleotide sequence encoding the bait region of the A2M protein.Similarly, “A2M cDNA”, “A2M coding sequence” or “A2M coding nucleicacid”, when used with reference to SEQ ID NOs: 1 or 2, means the nucleicacid sequences of SEQ ID NOs: 1 or 2, or fragments thereof, as well asnucleic acid variants which include one or more mutations, insertions,deletions, or a combination thereof. The A2M polynucleotides, orfragments thereof, can be manipulated using conventional techniques inmolecular biology so as to create variant A2M recombinant polynucleotideconstructs, encoding the variant A2M polypeptides that express variantA2M polypeptides. Variant A2M polynucleotides include nucleotidesequences having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NOs: 1 and2. A2M coding sequences includes nucleotide sequences having at least99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or85% sequence identity to any one of SEQ ID NOs: 1 and 2.

In one aspect, provided herein is a variant A2M polynucleotidenucleotide composition. Numerous polynucleotide sequences encodingwild-type A2M proteins from various organisms have been determined AnyA2M DNA sequence identified can be subsequently obtained by chemicalsynthesis and/or a polymerase chain reaction (PCR) technique such asoverlap extension method. For a short sequence, completely de novosynthesis may be sufficient; whereas further isolation of full lengthcoding sequence from a human cDNA or genomic library using a syntheticprobe may be necessary to obtain a larger gene. Alternatively, a nucleicacid sequence encoding an A2M polypeptide can be isolated from a humancDNA or genomic DNA library using standard cloning techniques such aspolymerase chain reaction (PCR), where homology-based primers can oftenbe derived from a known nucleic acid sequence encoding an A2Mpolypeptide.

cDNA libraries suitable for obtaining a coding sequence for a wild-typeA2M polypeptide can be obtained commercially or can be constructed. Thegeneral methods of isolating mRNA, making cDNA by reverse transcription,ligating cDNA into a recombinant vector, transfecting into a recombinanthost for propagation, screening, and cloning are well known. Uponobtaining an amplified segment of nucleotide sequence by PCR, thesegment can be further used as a probe to isolate the full-lengthpolynucleotide sequence encoding the wild-type A2M protein from the cDNAlibrary. A similar procedure can be followed to obtain a full lengthsequence encoding a wild-type A2M protein from a human genomic library.Human genomic libraries are commercially available or can be constructedaccording to various art-recognized methods. In general, to construct agenomic library, the DNA is first extracted from a tissue where apeptide is likely found. The DNA is then either mechanically sheared orenzymatically digested to yield fragments of about 12-20 kb in length.The fragments are subsequently separated by gradient centrifugation frompolynucleotide fragments of undesired sizes and are inserted inbacteriophage λ vectors. These vectors and phages are packaged in vitro.Recombinant phages are analyzed by plaque hybridization.

Based on sequence homology, degenerate oligonucleotides can be designedas primer sets and PCR can be performed under suitable conditions toamplify a segment of nucleotide sequence from a cDNA or genomic library.Using the amplified segment as a probe, the full-length nucleic acidencoding a wild-type A2M protein can be obtained

Upon acquiring a nucleic acid sequence encoding a wild-type A2M protein,the coding sequence can be subcloned into a vector, for instance, anexpression vector, so that a recombinant wild-type A2M protein can beexpressed mutated into a variant A2M polypeptide of the inventionproduced from the resulting construct. Further modifications to thewild-type A2M protein coding sequence, for example, nucleotidesubstitutions, may be subsequently made to alter the bait region of theA2M protein.

The present invention further provides isolated polypeptides encoded bythe polynucleotides, or fragments thereof, of the present invention orby degenerate variants of the polynucleotides, or fragments thereof, ofthe present invention. Preferred polynucleotides, or fragments thereof,of the present invention are the ORFs that encode A2M variants.

A variant A2M polynucleotide can be made by mutating the polynucleotidesequence encoding a wild-type A2M protein. This can be achieved by usingany known mutagenesis methods. Exemplary modifications to a wild-typeA2M polynucleotide for accepting variant bait regions described hereininclude those in SEQ ID NO 2. Exemplary modifications to an A2Mnucleotide include inserting or substituting a nucleotide sequenceencoding a variant bait region of SEQ ID NO 5-66 into the wild-type A2Mpolynucleotide sequence of SEQ ID NO: 1 and the variant A2M acceptorpolynucleotide sequence of SEQ ID NO 2. Mutagenesis procedures can beused separately or in combination to produce variants of a set ofnucleic acids, and hence variants of encoded polypeptides. Kits formutagenesis are commercially available.

In one aspect, provided herein are methods of making any of the variantA2M polynucleotides. A method of making a variant A2M polynucleotide cancomprise inserting or substituting a variant bait region into awild-type A2M polynucleotide sequence or substantially similar sequence.The substantially similar sequence can be SEQ ID NO 2. One aspect of theinvention is a method for making a variant A2M polynucleotidecomprising: a) providing a vector containing a variant A2Mpolynucleotide comprising a sequence of SEQ ID NO 2; b) digesting thevector containing a variant A2M polynucleotide with restrictionendonucleases to form a linear vector; c) ligating one end of the one ormore polynucleotides encoding one or more of the non-natural baitregions of SEQ ID NOs 5-66 to one end of the linear vector; and d)ligating the other end of the one or more polynucleotides encoding oneor more of the non-natural bait regions of SEQ ID NOs 5-66 to the otherend of the linear vector, thereby forming a vector containing a variantA2M polynucleotide comprising the non-natural bait regions of SEQ ID NOs5-66.

Protein Production

A variety of methodologies known in the art can be utilized to obtainany one of the isolated A2M variant proteins of the present invention.At the simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. Such polypeptides can besynthesized with or without a methionine on the amino terminusChemically synthesized polypeptides can be oxidized using methods setforth in these references to form disulfide bridges. The syntheticallyconstructed A2M variant sequences, by virtue of sharing primary,secondary or tertiary structural and/or conformational characteristicswith A2M variants can possess biological properties in common therewith,including protease inhibitory activity. This technique can beparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the A2M variants. Thus, they can be employed asbiologically active or immunological substitutes for natural, purifiedA2M variants in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The A2M variant polypeptides of the present invention can alternativelybe purified from cells which have been altered to express the desiredA2M variant. As used herein, a cell can be said to be altered to expressa desired A2M variant polypeptide or protein when the cell, throughgenetic manipulation, can be made to produce a A2M variant polypeptidewhich it normally does not produce or which the cell normally producesat a lower level. One skilled in the art can readily adapt proceduresfor introducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the A2M variant polypeptides of the present invention.

A variant A2M polypeptide can be a recombinant protein, or fragmentsthereof, and can be produced in a host cell or in vitro system.Recombinant polypeptides and protein promoters can be inserted in such amanner that it can be operatively produced in a host cell, for example,a bacterial culture or lower eukaryotes such as yeast or insects or inprokaryotes or any host know in the art. A variant A2M recombinantprotein can be produced in a bacterium, yeast, fungi, insect, ormammalian host cell, or a cell free system. For example, a variant A2Mpolypeptide can be produced in Escherichia coli, Bacillus subtilis,Salmonella typhimurium, Corynebacterium, Saccharomyces cerevisiae,Schizosaccharomyces pombe Kluyveromyces strains, Candida, Pichiapastoris, baculovirus-infected insect cells, or mammalian cells such asCOS cells, BHK cells, 293 cells, 3T3 cells, NS0 hybridoma cells, babyhamster kidney (BHK) cells, PER.C6™ human cells, HEK293 cells orCricetulus griseus (CHO) cells. A variant A2M polypeptide can beproduced by transient expression, stable cell lines, BacMam-mediatedtransient transduction, or cell-free protein production.

The variant A2M polypeptides can also be produced by operably linkingthe isolated variant A2M polynucleotides to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the variant A2M nucleotide sequence of interest can be ligatedto an adenovirus transcription/translation control complex, for example,the late promoter and tripartite leader sequence. This chimeric gene canthen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genomecan result in a recombinant virus that is viable and capable ofexpressing the variant A2M gene product in infected hosts. Specificinitiation signals can also be required for efficient translation ofinserted nucleotide sequences. These signals include the ATG initiationcodon and adjacent sequences. In cases where an entire variant A2M geneor cDNA, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, for example, a pJ608mammalian expression vector no additional translational control signalsare needed. Exogenous translational control signals, such as the ATGinitiation codon, can be provided.

Host cells can be genetically engineered to contain the variant A2Mpolynucleotides of the invention. For example, such host cells cancontain variant A2M polynucleotides introduced into the host cell usingknown transformation, transfection or infection methods. As used herein,a cell capable of expressing a variant A2M polynucleotide can be“transformed.” The variant A2M polypeptides of the invention can beprepared by culturing transformed host cells under culture conditionssuitable to express the recombinant protein. Any procedure forintroducing foreign nucleotide sequences into host cells may be used.Non-limiting examples include the use of calcium phosphate transfection,transfection, DEAE, dextran-mediated transfection, microinjection,lipofection, polybrene, protoplast fusion, electroporation (Davis, L. etal., Basic Methods in Molecular Biology (1986)), liposomes,microinjection, plasma vectors, viral vectors, and any other well-knownmethods for introducing cloned genomic DNA, cDNA, synthetic DNA, orother foreign genetic material into a host cell. A genetic engineeringprocedure capable of successfully introducing at least one gene into thehost cell capable of expressing the variant A2M polynucleotide can beused.

The present invention still further provides host cells engineered toexpress the variant A2M polynucleotides of the invention, wherein thevariant A2M polynucleotides are operative with a regulatory sequenceheterologous to the host cell which drives expression of the variant A2Mpolynucleotides in the cell. Knowledge of A2M-like DNA allows formodification of cells to permit, or increase, expression of A2M-likepolypeptide. Cells can be modified, for example, by homologousrecombination, to provide increased variant A2M polypeptide expressionby replacing, in whole or in part, the naturally occurring A2M derivedfrom the SV40 viral genome, for example, SV40 macroglobulin-likepromoter with all or part of a heterologous promoter so that the cells'variant A2M sites can be used to provide the required non-transcribedpolypeptide and can be expressed at higher levels.

For long-term, high-yield production of recombinant variant A2Mpolypeptides, stable expression is preferred. For example, cell linesthat stably express the variant A2M sequences described herein can beengineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells are allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn are cloned and expandedinto cell lines. This method is advantageously used to engineer celllines which express the variant A2M gene product. Such engineered celllines are particularly useful in screening and evaluation of compoundsthat affect the endogenous activity of the variant A2M gene product. Anumber of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase, and adenine phosphoribosyltransferase genescan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate; gpt,which confers resistance to mycophenolic acid; neo, which confersresistance to the aminoglycoside G-418; and hygro, which confersresistance to hygromycin.

Variant A2M polynucleotide sequences can be engineered so as to modifyprocessing or expression of the protein. For example, and not by way oflimitation, the variant A2M polynucleotides can be combined with apromoter sequence and/or ribosome binding site, or a signal sequence canbe inserted upstream of variant A2M polynucleotide sequences to permitsecretion of the variant A2M polypeptide and thereby facilitateharvesting or bioavailability. Additionally, a variant A2Mpolynucleotide can be mutated in vitro or in vivo, to create and/ordestroy translation, initiation, and/or termination sequences, or tocreate variations in coding regions and/or form new restriction sites ordestroy preexisting ones, or to facilitate further in vitromodification. Any technique for mutagenesis known in the art can beused, including but not limited to, in vitro site-directed mutagenesis.

Further, nucleic acids encoding other proteins or domains of otherproteins can be joined to nucleic acids encoding variant A2Mpolypeptides or fragments thereof so as to create a fusion protein.Nucleotides encoding fusion proteins can include, but are not limitedto, a full length variant or wild-type A2M protein, a truncated variantor wild-type A2M protein or a peptide fragment of a variant or wild typeA2M protein fused to an unrelated protein or peptide, such as forexample, a transmembrane sequence, which anchors the A2M peptidefragment to the cell membrane; an Ig Fc domain which increases thestability and half-life of the resulting fusion protein; maltose bindingprotein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), aHis tag, an enzyme, fluorescent protein, luminescent protein which canbe used as a marker, for example, an A2M-Green Fluorescent Proteinfusion protein. The fusion proteins can be used for affinitypurification.

The variant A2M nucleic acids and polypeptides can also be expressed inorganisms so as to create a transgenic organism. Desirable transgenicplant systems having one or more of these sequences include Arabadopsis,Maize, and Chlamydomonas. Desirable insect systems having one or more ofthe variant A2M polynucleotides and/or polypeptides include, forexample, D. melanogaster and C. elegans Animals of any species,including, but not limited to, amphibians, reptiles, birds, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees can be used togenerate variant A2M containing transgenic animals. Transgenic organismsdesirably exhibit germline transfer of variant A2M nucleic acids andpolypeptides described herein.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. Thesynthetically constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins can possess biological properties incommon therewith, including protein activity. This technique can beparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. Thus, they can be employed asbiologically active or immunological substitutes for natural, purifiedproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies. The polypeptides andproteins of the present invention can alternatively be purified fromcells which have been altered to express the desired polypeptide orprotein. As used herein, a cell can be said to be altered to express adesired polypeptide or protein when the cell, through geneticmanipulation, can be made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells in a suitable culture medium,and purifying the protein from the cells or the culture in which thecells are grown. For example, the methods can include a process forproducing a polypeptide in which a host cell containing a suitableexpression vector that includes a polynucleotide of the invention can becultured under conditions that allow expression of the encodedpolypeptide. The polypeptide can be recovered from the culture,conveniently from the culture medium, or from a lysate prepared from thehost cells and further purified. Preferred embodiments include those inwhich the protein produced by such process can be a full length ormature form of the protein, such as A2M. In an alternative method, thepolypeptide or protein can be purified from bacterial cells whichnaturally produce the polypeptide or protein. One skilled in the art canreadily follow known methods for isolating polypeptides and proteins inorder to obtain one of the isolated polypeptides or proteins of thepresent invention. These include, but are not limited to,immunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography, and immuno-affinity chromatography. See, e.g., Scopes,Protein Purification: Principles and Practice, Springer-Verlag (1994);Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel etal., Current Protocols in Molecular Biology. Polypeptide fragments thatretain biological or immunological activity include fragments comprisinggreater than about 100 amino acids, or greater than about 200 aminoacids, and fragments that encode specific protein domains. The purifiedpolypeptides can be used in in vitro binding assays which are well knownin the art to identify molecules which bind to the polypeptides. Thesemolecules include but are not limited to, for example, small molecules,molecules from combinatorial libraries, antibodies or other proteins.The molecules identified in a binding assay can then be tested forantagonist or agonist activity in in vivo tissue culture or animalmodels that are well known in the art. In brief, the molecules can betitrated into a plurality of cell cultures or animals and then testedfor either cell or animal death or prolonged survival of the animal orcells.

The resulting expressed variant A2M polypeptides can then be purifiedfrom a culture, for example, from culture medium or cell extracts, usingknown purification processes, such as affinity chromatography, gelfiltration, and ion exchange chromatography. The purification of thevariant A2M polypeptides can also include an affinity column containingagents which will bind to the protein; one or more column steps oversuch affinity resins as concanavalin A-agarose, heparin-Toyopearl™ orCibacron blue 3GA Sepharose™; one or more steps involving hydrophobicinteraction chromatography using such resins as phenyl ether, butylether, or propyl ether; or immunoaffinity chromatography. Alternatively,the protein of the invention can also be expressed in a form which willfacilitate purification. For example, a protein can be expressed as afusion protein, such as those of maltose binding protein (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag.Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteincan also be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“FLAG®”)is commercially available from Kodak (New Haven, Conn.). Finally, one ormore reverse-phase high performance liquid chromatography (RP-HPLC)steps employing hydrophobic RP-HPLC media, for example, silica gelhaving pendant methyl or other aliphatic groups, can be employed tofurther purify the protein. Any combination of the foregoingpurification procedures can also be employed to provide a substantiallyhomogeneous isolated or purified recombinant variant A2M polypeptide.The variant A2M polypeptides purified can be substantially free of othermammalian proteins and can be defined in accordance with the presentinvention as an “isolated protein.”

Agents for Inhibition of FAC Formation and Treatment of Chronic Wounds

Also provided herein are methods to inhibit the one or more steps of thefibronectin-aggrecan complex formation cycle (FACC) in a human with acondition or disease, such as a chronic wound. An agent can beadministered to a subject with a condition or disease. An agent can bewild-type A2M protein or a composition described herein, such as apurified form of A2M, or an A2M enriched sample, or a variant A2Mpolypeptide as described herein. An agent can be an agent that is not apurified form of A2M concentrated from autologous blood. An agent can bean inhibitor or an antagonist. An inhibitor or antagonist can be acompound or composition that directly or indirectly, partially ortotally blocks activity, decreases, prevents, delays activation,inactivates, desensitizes, or down regulates the activity or expressionof a target biomarker. Antagonists can be, for example, polypeptides,such as antibodies, and soluble receptors, as well as nucleic acids suchas siRNA or antisense RNA, as well as naturally occurring and syntheticbiomarker antagonists, including small chemical molecules.

An agent can be compound that has a pharmacological activity. Agents caninclude compositions described herein or compounds that are known drugs,compounds for which pharmacological activity has been identified butthat are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity. An agent can be organic or inorganic chemicalsuch a peptide, protein, including antibodies, small molecules andnatural products.

An agent can comprise an antibody. An antibody can be a polypeptidecomprising a framework region from an immunoglobulin gene or fragmentsthereof that specifically binds and recognizes an antigen. Therecognized immunoglobulin genes can include the kappa, lambda, alpha,gamma, delta, epsilon, and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Light chains can beclassified as either kappa or lambda. Heavy chains can be classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Anantibody can encompass plural referents unless the context clearlyindicates otherwise. In some instances a plurality of the antibodies canbelong to the same antibody species, e.g., in the case of monoclonalantibodies, while in some cases different antibodies species areencompassed the by phrase “an antibody”, e.g., a polyclonal antibodies.An exemplary immunoglobulin (antibody) structural unit can comprise atetramer. Each tetramer can be composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain can definea variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (V,)and variable heavy chain (VH) can refer to these light and heavy chainsrespectively. Antibodies can exist, e.g., as intact immunoglobulins oras a number of well-characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′2, a dimerof Fab which itself is a light chain joined to VH-CH1 by a disulfidebond. The F(ab)′2 may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′2dimer into an Fab′ monomer. The Fab′ monomer can be a Fab with part ofthe hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. An antibody can also be an antibody fragment eitherproduced by the modification of whole antibodies, or those synthesizedde novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty etal., Nature 348:552-554 (1990)). When referring to treatment methods,antibodies that are chimeric, human, humanized or otherwise specific tothe species to be treated can be used.

An agent can be an antibody that binds to the FAC but not to theindividual components of the complex separately. An agent can comprisean antibody that binds to aggrecan or any variation thereof, therebyinhibiting formation of the FAC complex. An agent can comprise anantibody, such as a monoclonal antibody, that binds to aggrecan G3lectin domain. The antibody can bind to aggrecan G3 and prevent theformation of FAC and inflammation. An agent can comprise an antibodythat binds to fibronectin or any variant thereof, thereby inhibitingformation of the FAC complex. An agent can comprise an antibody thatbinds to a PAMP receptor recognition domain of aggrecan, a DAMP receptorrecognition domain of aggrecan, or both, thereby inhibiting activationof monocytes and other cells. Other cells can be macrophages,fibroblast, T-cells, B-cells, neutrophils, platelets, synoviocytes,chondrocytes and other cells involved in inflammation. An agent cancomprise an antibody that binds to a PAMP receptor recognition domain offibronectin, a DAMP receptor recognition domain of fibronectin, or both,thereby inhibiting activation of monocytes and other cells.

An agent that prevents or inhibits FAC formation can be a recombinantaggrecan G3 domain, wherein the domain contains the aggrecan G3 lectindomain and competitively binds to fibronectin; wherein the domain lacksthe Pathogen Associated Molecular Patterns (PAMP) and the DamageAssociated Molecular Patterns (DAMP) receptor recognition domainsRecombinant aggrecan G3 lectin domain can competitively bind tofibronectin. A recombinant aggrecan G3 domain can lack the cellactivation domain and can slow down, inhibit, or prevent FAC formationand inflammation.

An agent that prevents or inhibits FAC formation can be a wild-type A2Mprotein or a recombinant fibronectin fragment, wherein the fragmentcomprises a G3 binding domain and binds to aggrecan, wherein the G3binding domain the PAMP, receptor recognition domain, the DAMP receptorrecognition domain, or both. A recombinant fibronectin fragment cancompetitively bind to aggrecan.

An agent that prevents or inhibits FAC formation can be a soluble formof the PAMP receptor or DAMP receptor that binds to the PAMP domain ofaggrecan G3, the DAMP domain of aggrecan G3, or both, thereby inhibitingactivation of monocytes and other cells to produce proinflammatorycytokines, chemokines, proteases, or any combination thereof.

An agent that prevents or inhibits FAC formation can be a soluble formof the PAMP receptor or DAMP receptor that binds to the PAMP domain offibronectin, the DAMP domain of fibronectin, or both, thereby inhibitingactivation of monocytes and other cells to produce proinflammatorycytokines, chemokines, proteases, or any combination thereof. An agentcan be an inhibitor of fibroblast cells and can inhibit production ofincreased levels fibronectin, recruitment of other fibroblast cells, orboth.

An agent that prevents or inhibits FAC formation can be a smallmolecule. A small molecule can be identified using one or morehigh-throughput screening methods. A small molecule can inhibit FACformation, inhibit activation of monocytes; inhibit increased productionof fibronectin; inhibit recruitment of fibroblast cells; or bind to theDAMP domain of fibronectin, bind to the DAMP domain of aggrecan G3, bindto the PAMP domain of fibronectin, or bind to the PAMP domain ofaggrecan G3, thereby inhibiting activation of cells to produceproinflammatory cytokines, chemokines, proteases, or any combinationthereof. In A small molecule can inhibit FAC formation by competitivelybinding to fibronectin or aggrecan. In some embodiments, the smallmolecule binds to the FAC complex and resulting in dissociation ordegradation of the FAC complex Inhibiting the formation of thefibronectin-aggrecan complex (FAC) can comprise inhibiting one or moresteps in FAC formation or a step in the FAC formation cycle (FACC).

One or more steps in FAC formation or the FACC can comprise productionof fibronectin in the ECM, production of proteases and metalloproteases,production of inflammatory cytokines and chemokines, degradation ofaggrecan in cartilage, or increasing the aggrecan G3 domain fragmentconcentration.

In any of the methods herein, an agent can be a medicament used to treatchronic wounds such as pressure ulcers, venous ulcers, stasis ulcers,venous stasis ulcers, diabetic foot ulcers, arterial insufficiencyulcers or any combination thereof. Thus, one can administer to asubject, along with a composition comprising an elevated concentrationof A2M, a variant A2M polypeptide, or a wild-type A2M protein, aneffective amount of one or more other medicament (where a compositioncomprising an elevated concentration of A2M or variant A2Mpolynucleotide (e.g., compositions described herein) can be a firstmedicament). The one or more other medicaments can include, for example,an immunosuppressive agent, a cytokine antagonist such as a cytokineantibody, an integrin antagonist (e.g., antibody), a corticosteroid, orany combination thereof. The type of such second medicament can dependon various factors, including the type of chronic wound, extent of thewound, the severity of the wound, the condition and age of the subject,the type and dose of the first medicament employed, etc. Examples ofsuch additional medicaments include an immunosuppressive agent (such asmitoxantrone (NOVANTRONE®), MTX, cyclophosphamide, chlorambucil,leflunomide, and azathioprine), intravenous immunoglobulin (gammaglobulin), lymphocyte-depleting therapy (e.g., mitoxantrone,cyclophosphamide, CAMPATH™ antibodies, anti-CD4, cladribine, apolypeptide construct with at least two domains comprising ade-immunized, autoreactive antigen or its fragment that can bespecifically recognized by the Ig receptors of autoreactive B-cells (WO2003/68822), total body irradiation, and bone marrow transplantation),integrin antagonist or antibody (e.g., an LFA-1 antibody such asefalizumab/RAPTIVA® commercially available from Genentech, or an alpha 4integrin antibody such as natalizumab/ANTEGREN® available from Biogen,or others as noted above), drugs that treat symptoms secondary orrelated to chronic wound healing such as those noted herein, steroidssuch as corticosteroid (e.g., prednisolone, methylprednisolone such asSOLU-MEDROL™ methylprednisolone sodium succinate for injection,prednisone such as low-dose prednisone, dexamethasone, orglucocorticoid, e.g., via injection, including systemic corticosteroidtherapy), nonlymphocyte-depleting immunosuppressive therapy (e.g., MMFor cyclosporine), a TNF-α inhibitor such as an antibody to TNF-α or itsreceptor or TNFR-Ig (e.g., etanercept), DMARD, NSAID, plasmapheresis orplasma exchange, trimethoprim-sulfamethoxazole (BACTRIM™, SEPTRA™), MMF,H2-blockers or proton-pump inhibitors (during the use of potentiallyulcerogenic immunosuppressive therapy), levothyroxine, cyclosporin A(e.g., SANDIMMUNE®), somatostatin analogue, a DMARD or NSAID, cytokine25 antagonist such as antibody, anti-metabolite, immunosuppressiveagent, rehabilitative surgery, radioiodine, thyroidectomy, anti-IL-6receptor antagonist/antibody (e.g., ACTEMRA™ (tocilizumab)), or anotherB-cell antagonist such as BR3-Fc, TACI-Ig, anti-BR3 antibody, anti-CD40receptor or anti-CD40 ligand (CD154), agent blocking CD4O-CD40 ligand,epratuzumab (anti-CD22 antibody), lumiliximab (anti-CD23 30 antibody),or anti-CD20 antibody such as rituximab or 2H7 antibody. Knowninhibitors such as chelators of known aggrecanases or MMP's can beadministered to a subject in need thereof in amount effective to inhibitor slow down the release of aggrecan fragments which in effect willreduce or eliminate the formation of the fibronectin aggrecan complexesthereby giving relief to the subject from the chronic wound.

Diagnostic Methods

Methods for detecting biomarkers, such as a wild-type A2M protein, toidentify sites in the subject that are a source of chronic wounds can beused to diagnose, or assist in the diagnosis be of, subjects withchronic wounds related to the anatomic structure and physiologicfunction of the wound. For example, the identification offibronectin-aggrecan complexes in a biological sample, such as abiological sample from the wound can be used to diagnose, or assist inthe diagnosis of a chronic wound.

The amount of a biomarker, such as A2M, that can indicate a specificlocation in the subject as a source of a chronic wound for a particularsubject can depend on numerous factors, including, but not limited to,the age, sex, medical history, etc., of the patient, the site that thebiological sample was extracted from, and the assay format used todetect the biomarker. In some embodiments, the level and/orconcentration of A2M in a biological sample may be quantified ordirectly compared with a control sample. In some embodiments, the leveland/or concentration of A2M in a biological sample may not be quantifiedor directly compared with a control sample, but can rather be detectedrelative to a “diagnostic absence” or “diagnostic presence” of A2M.

A “diagnostic absence” can refer to an amount and/or concentration ofA2M in a biological that indicates the absence or likelihood of theabsence of a chronic wound or inflammation causing pathology or injuryat the location from which the sample was taken. A diagnostic absencecan be detectable in a simple assay giving a positive or negativeresult. A positive or negative result can be determined based on theamount and/or concentration of A2M in the biological sample. Detectionof a level and/or concentration of A2M corresponding to a diagnosticabsence of A2M indicates the absence of a chronic wound-causingpathology or injury at the location from which the sample was taken. Insome embodiments, a diagnostic absence of A2M can be a concentration ofA2M in a biological sample from about 0-30 μg/ml. For example, adiagnostic absence of A2M can be a concentration of A2M in a biologicalsample from about 0-30 μg/ml, 0-25 μg/ml, 0-20 μg/ml, 0-15 μg/ml, 0-10μg/ml, 0-5 μg/ml, 5-30 μg/ml, 5-25 μg/ml, 5-20 μg/ml, 5-15 μg/ml, 5-10μg/ml, 10-30 μg/ml, 10-25 μg/ml, 10-20 μg/ml, 10-15 μg/ml, 15-30 μg/ml,15-25 μg/ml, 15-20 μg/ml, 20-30 μg/ml, or 20-25 μg/ml. In someembodiments, a diagnostic absence of A2M can be a concentration of A2Min a biological sample from about 0-40 μg/ml. For example, a diagnosticabsence of A2M can be a concentration of A2M in a biological sample fromabout 0-40 μg/ml, 5-40 μg/ml, 10-40 μg/ml, 15-40 μg/ml, 20-40 μg/ml,25-40 μg/ml, 30-40 μg/ml, or 35-40 μg/ml.

In some embodiments, a diagnostic absence of A2M in a biological samplecan be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1000 or more fold lower than a controlsample.

A “diagnostic presence” can refer to an amount and/or concentration ofA2M in a biological that indicates the presence or likelihood of thepresence of a chronic wound or inflammation causing pathology or injuryat the location from which the sample was taken. A diagnostic presencecan be detectable in a simple assay giving a positive or negativeresult. A positive or negative result can be determined based on theamount and/or concentration of A2M in the biological sample. Detectionof a level and/or concentration of A2M corresponding to a diagnosticpresence of A2M indicates the presence of a chronic wound-causingpathology or injury at the location from which the sample was taken. Insome embodiments, a diagnostic presence of A2M can be a concentration ofA2M in a biological sample of at least about 31 μg/ml, 32 μg/ml, 33μg/ml, 34 μg/ml, 35 μg/ml, 36 μg/ml, 37 μg/ml, 38 μg/ml, or 39 μg/ml. Insome embodiments, a diagnostic presence of A2M can be a concentration ofA2M in a biological sample of at least about 40 μg/ml. For example, adiagnostic presence of A2M can be a concentration of A2M in a biologicalsample of at least about 45 μg/ml, 50 μg/ml, 55 μg/ml, 60 μg/ml, 65μg/ml, 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100μg/ml, 110 μg/ml, 120 μg/ml, 130 μg/ml, 140 μg/ml, 145 μg/ml, 150 μg/ml,160 μg/ml, 170 μg/ml, 180 μg/ml, 190 μg/ml, 200 μg/ml, 220 μg/ml, 240μg/ml, 250 μg/ml, 260 μg/ml, 280 μg/ml, 300 μg/ml, 320 μg/ml, 340 μg/ml,360 μg/ml, 380 μg/ml, 400 μg/ml, 420 μg/ml, 440 μg/ml, 460 μg/ml, 480μg/ml, 500 μg/ml, or more.

In some embodiments, a diagnostic presence of A2M can be a concentrationof A2M in a biological sample from about 40-500 μg/ml. For example, adiagnostic presence of A2M can be a concentration of A2M in a biologicalsample from about 50-500 μg/ml, 60-500 μg/ml, 70-500 μg/ml, 80-500μg/ml, 90-500 μg/ml, 100-500 μg/ml, 125-500 μg/ml, 150-500 μg/ml,175-500, 200-500 μg/ml, 250-500 μg/ml, 300-500 μg/ml, 400-500 μg/ml,50-60 μg/ml, 50-70 μg/ml, 50-80 μg/ml, 50-90 μg/ml, 50-100 μg/ml, 50-125μg/ml, 50-150 μg/ml, 50-175 μg/ml, 50-200 μg/ml, 50-250 μg/ml, 50-300μg/ml, 50-400 μg/ml, 60-70 μg/ml, 60-80 μg/ml, 60-90 μg/ml, 60-100μg/ml, 60-125 μg/ml, 60-150 μg/ml, 60-175 μg/ml, 60-200 μg/ml, 60-250μg/ml, 60-300 μg/ml, 60-400 μg/ml, 70-80 μg/ml, 70-90 μg/ml, 70-100μg/ml, 70-125 μg/ml, 70-150 μg/ml, 70-175 μg/ml, 70-200 μg/ml, 70-250μg/ml, 70-300 μg/ml, 70-400 μg/ml, 80-90 μg/ml, 80-100 μg/ml, 80-125μg/ml, 80-150 μg/ml, 80-175 μg/ml, 80-200 μg/ml, 80-250 μg/ml, 80-300μg/ml, 80-400 μg/ml, 90-100 μg/ml, 90-125 μg/ml, 90-150 μg/ml, 90-175μg/ml, 90-200 μg/ml, 90-250 μg/ml, 90-300 μg/ml, 90-400 μg/ml, 100-125μg/ml, 100-150 μg/ml, 100-175 μg/ml, 100-200 μg/ml, 100-250 μg/ml,100-300 μg/ml, 100-400 μg/ml, 125-150 μg/ml, 125-175 μg/ml, 125-200μg/ml, 125-250 μg/ml, 125-300 μg/ml, 125-400 μg/ml, 150-175 μg/ml,150-200 μg/ml, 150-250 μg/ml, 150-300 μg/ml, 150-400 μg/ml, 175-200μg/ml, 175-250 μg/ml, 175-300 μg/ml, 175-400 μg/ml, 200-250 μg/ml,200-300 μg/ml, 200-400 μg/ml, 250-300 μg/ml, 250-400 μg/ml, or 300-400μg/ml.

In some embodiments, a diagnostic presence of A2M in a biological samplecan be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1000 or more fold higher than a controlsample.

The disclosed methods can be used regardless of whether A2M is normallypresent, or expected to be present, in a particular control sample. Forexample, A2M may not be detectable in certain normal wound samples usinga particular assay, resulting in a complete absence of A2M complexes ina control biological sample. For example, A2M cannot be detectable incertain normal wound samples (such as, for example, in a synovial fluidsample) using a particular assay, resulting in a complete absence of A2Min a control biological sample. For such biological samples, adiagnostic presence can refer to any detectable amount of A2M using thatsame assay. In other instances, however, there can be a detectable levelof A2M present in normal or control samples and a diagnostic absencerepresents a level that can be lower than the normal level, preferablyrepresenting a statistically significant decrease over the normal level.

Control samples can be samples that are taken from an individual or agroup of individuals not experiencing inflammation or a chronic wound,such as pressure ulcers, venous ulcers, stasis ulcers, venous stasisulcers, diabetic foot ulcers, arterial insufficiency ulcers or anycombination thereof. Alternatively, control samples can be obtained froma source not suspected to be a source of a chronic wound orinflammation, such as a level of the wound not suspected to be a sourceof chronic wound. For example, in a subject experiencing a wound, thecontrol sample can be obtained from an unaffected or asymptomatic regionof the same patient. Control samples can be samples that are taken froman individual or a group of individuals not experiencing a chronicwound. Alternatively, control samples can be obtained from unaffected orasymptomatic wounds from the subject being tested.

The level of a biomarker, such as A2M, need not be quantified for adiagnostic absence or presence to be detected. Rather, any method ofdetermining whether A2M is present at levels lower or higher than in anormal or control can be used. In addition, a diagnostic absence orpresence does not refer to any absolute quantity of A2M, but rather toan amount that, depending on the biological sample, assay conditions,medical condition of the patient, etc., can be sufficient to distinguishthe level in an affected patient from a normal or control patient.

The presence, absence or level of A2M present at a particular levelwithin the wound can be used to diagnose, or assist in the diagnosis beof, a particular type of chronic wounds such as pressure ulcers, venousulcers, stasis ulcers, venous stasis ulcers, diabetic foot ulcers,arterial insufficiency ulcers or any combination thereof. Additionally,or alternatively, the presence, absence, or level of A2M in a woundsample can be used to distinguish wounds that results from woundpathology or injury originating from another source, such asnon-chronic-wound.

The presence, absence, or change over time in the level of A2M in abiological sample can be used to designate a patient as candidate for aparticular treatment. A sample obtained from the patient can be analyzedfor the presence or absence of A2M. The patient can be selected fortreatment if A2M is not detected in the sample. The type of treatmentcan be then tailored to the severity of the condition as determined bythe presence, absence, or level of A2M.

The level A2M present at a specific site can also be useful to determinea prognosis for the subject being tested. For example, the level of A2Mpresent in a sample can indicate the extent of an acute injury to thesubject and can assist a practitioner in determining to what extentsuccessful repair or healing of the injury or pathology can be achieved.

Methods for Detecting A2M to Identify Wounds as Sites for TreatingChronic Wounds can be Used to Diagnose, or Assist in the Diagnosis of,Subjects

Detection of A2M can be used alone, or in combination with otherdiagnostic approaches to diagnose chronic wounds. Exemplary diagnosticapproaches include, but are not limited to, medical history and physicalexamination, x-ray radiography, MRI and intra-articular injection. Thepresence of A2M can however be used to diagnose injury and administertreatment at a particular location irrespective of whether injury wasdetectable by other methods, e.g., an MRI. The patient will typically betreated by administration of a therapeutic agent to the site of injuryor pathology, i.e., the site of presence of A2M.

The diagnostic methods of the present invention can includedetermination of the expression levels of a set of nucleic acidmolecules comprising polynucleotide sequences coding for a proteinmarker. The diagnostic methods of the present invention can include thedetermination of expression levels of a plurality (i.e., one or more,e.g., at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10 or more) of polypeptides ina biological sample obtained from a subject. Determination of proteinexpression levels in the practice of the inventive methods can beperformed by any suitable method (see, for example, E. Harlow and A.Lane, “Antibodies: A Laboratories Manual”, 1988, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.).

The disclosed methods can also be used to assess the efficacy of atreatment or a course of treatment. For example, in a patient with woundtesting positive for a diagnostic positive of A2M, such as wild-type A2Mprotein, indicative of a chronic wound, the efficacy of a chronic woundtreatment can be assessed by monitoring, over time, the levels of A2M. Adecrease in the levels of A2M in a biological sample taken from apatient following a treatment, compared to a level in a sample takenfrom the same patient before, or earlier in, the treatment, can indicateefficacious treatment. An increase or lack of change in the levels ofA2M in a biological sample taken from a patient following a treatment,compared to a level in a sample taken from the same patient before, orearlier in, the treatment, can indicate a non-efficacious treatment.

Inflammation biomarkers for diagnostic methods can be A2M, chemokines,cytokines, fibronectin, or aggrecan polypeptides in any ratio, inaddition to other inflammatory mediators, extracellular matrix moleculesor their breakdown products, signal transduction mediators, proteasesand their inhibitors, and neurotransmitter receptors including, but notlimited to IL-6, Prostaglandin E2, NO, IFN gamma, SHT, RANTES, MIP-1a,MCP-1, IL-1ra, TNF-α, Procollagens, CTX II, ARGS, aggrecan fragments,fibronectin fragments, FAC, COMP, CS 846, chondroitin fragments, sRAGE,MMP-3, MMP-13 and other MMPs, ADAMTS-4, aggrecanases, NF-kappa-B, p38MAP kinase, DR5/DcR2. The biomarkers can include full lengthpolypeptides or can include fragments of polypeptides.

Any known method for detecting the presence of polypeptides in abiological sample can be used to qualitatively or quantitatively detectthe presence of A2M in biological samples, such as chronic woundsamples. Suitable methods include, but are not limited to,chromatographic methods, selective binding assays, mass spectrometry,spectrophotometry, or combinations thereof.

Exemplary binding assays include immunoassays, such as enzyme-linkedimmunosorbent assays Immunoassays can be used to qualitatively orquantitatively analyze a sample for the presence of A2M. A generaloverview of the applicable technology can be found in a number ofreadily available manuals, e.g., Harlow & Lane, Cold Spring HarborLaboratory Press, Using Antibodies: A Laboratory Manual (1999).

The disclosed methods and kits can utilize selective binding partners ofinflammation biomarkers to identify their presence or determine theirlevels in samples from the wound. The selective binding partners can beantibodies, or other biomolecules that specifically bind to A2M, orfragments or complexes thereof.

Monoclonal or polyclonal antibodies can be used. The antibodies can beany known in the art, including commercially available antibodies. It iswell known to those of skill in the art that the type, source and otheraspects of an antibody to be used can be a consideration to be made inlight of the assay in which the antibody can be used. In some instances,antibodies that will recognize its antigen target (for instance, anepitope or multiple epitopes from A2M) on a Western blot might not beapplicable to all ELISA or ELISpot assay and vice versa.

Antibodies, antibody fragments, or single chain antibodies to be usedcan be produced using techniques for producing monoclonal or polyclonalantibodies that are well known in the art (see, e.g., Coligan, CurrentProtocols in Immunology (1991); Harlow & Lane, supra; Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986); and Kohler &Milstein, Nature 256: 495-497 (1975). Such techniques include antibodypreparation by selection of antibodies from libraries of recombinantantibodies in phage or similar vectors, as well as preparation ofpolyclonal and monoclonal antibodies by immunizing rabbits or mice (see,e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature341:544-546 (1989)).

A number of immunogens from A2M can be used to produce antibodiesspecifically reactive with A2M and fragments thereof. For example, arecombinant A2M or an antigenic fragment thereof, can be isolated usingmethods well known to those of skill in the art. Recombinant protein canbe expressed in eukaryotic or prokaryotic cells. Recombinant protein canbe the typically used immunogen for the production of monoclonal orpolyclonal antibodies. Alternatively, synthetic peptides derived fromthe known sequences A2M and conjugated to a carrier protein can be usedas an immunogen. Naturally-occurring protein can also be used either inpure or impure form. The product can be then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated, for subsequent use in immunoassays tomeasure the protein.

Antibodies that specifically bind to complexes containing A2M can beused as specific binding partners.

Non-antibody polypeptides can be used as specific binding agents for thedetection of A2M, or fragments or complexes thereof. A large number ofproteins that specifically bind to A2M are known in the art. Exemplaryproteins that can be used as selective binding partners of A2M include,but are not limited to soluble receptors, cytokines and growth factorsthat are known to bind A2M, modified proteases that can bind to A2M andnot trigger the conformation change.

Once selective binding partners are available, each specific biomarkercan be detected by a variety of selective binding assays, includingimmunoassay methods. For a review of immunological and immunoassayprocedures, see Basic and Clinical Immunology (Stites & Terr eds., 7thed. 1991). Moreover, the disclosed selective binding assays can beperformed in any of several configurations. Several immunoassayconfigurations are reviewed extensively in Enzyme Immunoassay (Maggio,ed., 1980).

Methods for detecting the presence and/or measuring a level of A2M in asample, can use specific binding partners A2M, or fragments or complexesthereof. The methods generally include contacting the sample withspecific binding partner for A2M, or fragments or complexes thereof,purifying a desired fraction from the sample, and detecting bindingbetween the specific binding partner and molecules of the sample.

Detection of specific binding of the specific binding partners withmolecules of the sample, when compared to a suitable control, can be anindication that biomarkers are present in the sample. A variety ofmethods to detect specific protein interactions are known in the art andcan be used in the method. Methods include competitive assays andnoncompetitive assays.

Suitable methods include, but are not limited to, Western blot,immunoprecipitation, ELISA and radio-immunoassays. Methods forperforming these and other suitable assays are known in the art. Ingeneral, the specific binding partner used to detect the biomarker willbe delectably labeled, either directly or indirectly. The chronic woundsample can be brought into contact with and immobilized on a solidsupport or carrier, such as a membrane (i.e. nitrocellulose) orpolystyrene or magnetic beads that can be capable of immobilizing cells,cell particles, or soluble proteins. The support can then be washed withsuitable buffers, followed by contacting with a detectably-labeledselective binding partner.

In specific binding assays, it can be desirable to minimize the amountof non-specific binding that occurs, particularly when the specificbinding partner can be attached to a substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused. In addition to, or in place of proteinaceous material, variousdetergents and/or salt can be incorporated into the immunoassay tominimize non-specific interactions. Throughout the assays, incubationand/or washing steps can be required after each combination of reagents.Incubation and washing times will depend upon several factors, includingthe assay format, the affinity of the specific binding partner for thebiomarker, the volume of solution, concentrations.

A positive control for an inflammation biomarker can be used in thedetection assays, for example to calibrate the detection assay. A2M canbe from different sources, for example from different species. A2M canbe recombinant, natural or a combination thereof.

In general, protein expression levels can be determined by contacting abiological sample isolated from a subject with binding agents for one ormore of the protein markers; detecting, in the sample, the levels ofpolypeptides that bind to the binding agents; and comparing the levelsof polypeptides in the sample with the levels of polypeptides in acontrol sample. A binding agent can be an entity or composition such asa polypeptide or antibody that specifically binds to a protein marker. Abinding agent can specifically bind to a polypeptide if it reacts and/orinteracts at a detectable level with the polypeptide, but does not reactand/or interact detectably with peptides containing unrelated sequencesor sequences of different polypeptides.

The binding agent can be a ribosome, with or without a peptidecomponent, an RNA molecule, or a polypeptide (e.g., a polypeptide thatcomprises a polypeptide sequence of a protein marker, a peptide variantthereof, or a non-peptide mimetic of such a sequence).

The binding agent can be an antibody specific for a protein marker ofthe invention. Suitable antibodies for use in the methods of the presentinvention include monoclonal and polyclonal antibodies, immunologicallyactive fragments (e.g., Fab or (Fab)₂ fragments), antibody heavy chains,humanized antibodies, antibody light chains, and chimeric antibodies.Antibodies, including monoclonal and polyclonal antibodies, fragmentsand chimeras, can be prepared using methods known in the art (see, forexample, R. G. Mage and E. Lamoyi, in “Monoclonal Antibody ProductionTechniques and Applications”, 1987, Marcel Dekker, Inc.: New York, pp.79-97; G. Kohler and C. Milstein, Nature, 1975, 256: 495-497; D. Kozboret al., J. Immunol. Methods, 1985, 81: 31-42; and R. J. Cote et al.,Proc. Natl. Acad. Sci. 1983, 80: 2026-203; R. A. Lerner, Nature, 1982,299: 593-596; A. C. Nairn et al., Nature, 1982, 299: 734-736; A. J.Czernik et al., Methods Enzymol. 1991, 201: 264-283; A. J. Czernik etal., Neuromethods: Regulatory Protein Modification: Techniques &Protocols, 1997, 30: 219-250; A. J. Czernik et al., Neuroprotocols,1995, 6: 56-61; H. Zhang et al., J. Biol. Chem. 2002, 277: 39379-39387;S. L. Morrison et al., Proc. Natl. Acad. Sci., 1984, 81: 6851-6855; M.S. Neuberger et al., Nature, 1984, 312: 604-608; S. Takeda et al.,Nature, 1985, 314: 452-454). Antibodies to be used in the methods of theinvention can be purified by methods well known in the art (see, forexample, S. A. Minden, “Monoclonal Antibody Purification”, 1996, IBCBiomedical Library Series: Southbridge, Mass.). For example, antibodiescan be affinity purified by passage over a column to which a proteinmarker or fragment thereof can be bound. The bound antibodies can thenbe eluted from the column using a buffer with a high salt concentration.

Instead of being prepared, antibodies to be used in the methods of thepresent invention can be obtained from scientific or commercial sources.

The binding agent can be directly or indirectly labeled with adetectable moiety. The role of a detectable agent can be to facilitatethe detection step of the diagnostic method by allowing visualization ofthe complex formed by binding of the binding agent to the protein marker(or analog or fragment thereof). The detectable agent can be selectedsuch that it generates a signal which can be measured and whoseintensity can be related or proportional to the amount of protein markerpresent in the sample being analyzed. Methods for labeling biologicalmolecules such as polypeptides and antibodies are well-known in the art(see, for example, “Affinity Techniques. Enzyme Purification B”, Methodsin Enzymol., 1974, Vol. 34, W. B. Jakoby and M. Wilneck (Eds.), AcademicPress: New York, N.Y.; and M. Wilchek and E. A. Bayer, Anal. Biochem.,1988, 171: 1-32).

Specific binding to an antibody, for example, when referring to aprotein or peptide, can be a binding reaction that can be determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies can bind to a particular protein or protein complexat least two times the background and do not substantially bind in asignificant amount to other proteins present in the sample. Specificbinding between a binding agent, e.g., an antibody and a protein, forinstance, a biomarker, can be the ability of a capture- ordetection-agent to preferentially bind to a particular antigen that canbe present in a mixture; e.g., a biological sample. In some embodiments,specific binding can refer to a dissociation constant (K_(D)) that canbe less than about 10⁻⁶ M; preferably, less than about 10^(−s) M; and,most preferably, less than about 10⁻⁹ M.

Specific binding assays, including immunoassays, can use a labelingagent to specifically bind to and allow for the detection of the complexformed by the specific binding partner and the detected analyte. A labelor detectable moiety can be a composition detectable by spectroscopic,photochemical, biochemical, radiographic, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The labels can be incorporated into nucleicacids, proteins and antibodies at any position. Any method known in theart for conjugating the antibody to the label can be employed, e.g.,using methods described in Hermanson, Bioconjugate Techniques 1996,Academic Press, Inc., San Diego.

The labeling agent can be a part of the specific binding partner used todetect the analyte. Alternatively, the labeling agent can be a thirdmoiety, such a secondary antibody, which specifically binds to thecomplex formed by the specific binding partner and the detected analyte.Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagent. These proteins exhibit a strong affinity for immunoglobulinconstant regions from a variety of species (see, e.g., Kronval et al.,J. Immunol. 111: 1401-1406 (1973); Akerstrom et al., J. Immunol.135:2589-2542 (1985)). The labeling agent can be modified with adetectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well-known to those skilled in the art.

The detectable label can be any material having a detectable physical orchemical property. Many useful detectable labels are known in the artand include any label that can be detectable by spectroscopic,photochemical, biochemical, immunochemical, radiographic, electrical,optical or chemical means. The choice of label can depend on thesensitivity required, the ease of conjugation with the compound,stability requirements, available instrumentation, and disposalprovisions. Useful labels include magnetic beads (e.g., DYNABEADS®),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) can be covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which can be either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize the biomarkers, or secondary antibodies that recognizethe antibodies to the biomarkers.

The molecules can also be conjugated directly to signal generatingcompounds, for example, by conjugation with an enzyme or fluorophore.Enzymes that can be used as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Exemplary fluorescent compounds include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and μM belliferone. Exemplary chemiluminescentcompounds include, but are not limited to, luciferin and2,3-dihydrophthalazinediones. Means of detecting labels are well knownto those of skill in the art.

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides, fluorescent dyes,chemiluminescent agents, microparticles (such as, for example, quantumdots, nanocrystals, phosphors and the like), enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the binding agents (e.g., antibodies) can beimmobilized on a carrier or support (e.g., a bead, a magnetic particle,a latex particle, a microtiter plate well, a cuvette, or other reactionvessel). Examples of suitable carrier or support materials includeagarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose,liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene,gabbros, filter paper, magnetite, ion-exchange resin, plastic film,plastic tube, glass, polyamine-methyl vinylether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, andthe like. Binding agents can be indirectly immobilized using secondbinding agents specific for the first binding agents (e.g., mouseantibodies specific for the protein markers can be immobilized usingsheep anti-mouse IgG Fc fragment specific antibody coated on the carrieror support).

Protein expression levels in the diagnostic methods of the presentinvention can be determined using immunoassays. Examples of such assaysare radioimmunoassays, enzyme immunoassays (e.g., ELISA),immunofluorescence immunoprecipitation, latex agglutination,hemagglutination, and histochemical tests, which are conventionalmethods well-known in the art. As will be appreciated by one skilled inthe art, the immunoassay can be competitive or noncompetitive. Methodsof detection and quantification of the signal generated by the complexformed by binding of the binding agent with the protein marker willdepend on the nature of the assay and of the detectable moiety (e.g.,fluorescent moiety). Alternatively, the protein expression levels can bedetermined using mass spectrometry based methods or image (including useof labeled ligand) based methods known in the art for the detection ofproteins. Other suitable methods include proteomics-based methods.

Determination of expression levels of nucleic acid molecules in thepractice of the inventive methods can be performed by any suitablemethod, including, but not limited to, Southern analysis, Northernanalysis, polymerase chain reaction (PCR) (see, for example, U.S. Pat.Nos. 4,683,195; 4,683,202, and 6,040,166; “PCR Protocols: A Guide toMethods and Applications”, Innis et al. (Eds.), 1990, Academic Press:New York), reverse transcriptase PCR(RT-PCT), anchored PCR, competitivePCR (see, for example, U.S. Pat. No. 5,747,251), rapid amplification ofcDNA ends (RACE) (see, for example, “Gene Cloning and Analysis: CurrentInnovations, 1997, pp. 99-115); ligase chain reaction (LCR) (see, forexample, EP 01 320308), one-sided PCR (Ohara et al., Proc. Natl. Acad.Sci., 1989, 86: 5673-5677), in situ hybridization, Taqman based assays(Holland et al., Proc. Natl. Acad. Sci., 1991, 88:7276-7280),differential display (see, for example, Liang et al., Nucl. Acid. Res.,1993, 21: 3269-3275) and other RNA fingerprinting techniques, nucleicacid sequence based amplification (NASBA) and other transcription basedamplification systems (see, for example, U.S. Pat. Nos. 5,409,818 and5,554,527), Qbeta Replicase, Strand Displacement Amplification (SDA),Repair Chain Reaction (RCR), nuclease protection assays,subtraction-based methods, Rapid-Scan™, and the like.

Nucleic acid probes for use in the detection of polynucleotide sequencesin biological samples can be constructed using conventional methodsknown in the art. Suitable probes can be based on nucleic acid sequencesencoding at least about 5 sequential amino acids from regions of nucleicacids encoding a protein marker, and preferably comprise about 15 toabout 50 nucleotides. A nucleic acid probe can be labeled with adetectable moiety, as mentioned above in the case of binding agents. Theassociation between the nucleic acid probe and detectable moiety can becovalent or non-covalent. Detectable moieties can be attached directlyto nucleic acid probes or indirectly through a linker (E. S. Mansfieldet al., Mol. Cell. Probes, 1995, 9: 145-156). Methods for labelingnucleic acid molecules are well known in the art (for a review oflabeling protocols, label detection techniques and recent developmentsin the field, see, for example, L. J. Kricka, Ann Clin. Biochem. 2002,39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001,1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35:135-153).

Nucleic acid probes can be used in hybridization techniques to detectpolynucleotides encoding the protein markers. The technique cangenerally involve contacting an incubating nucleic acid molecules in abiological sample obtained from a subject with the nucleic acid probesunder conditions such that specific hybridization takes place betweenthe nucleic acid probes and the complementary sequences in the nucleicacid molecules. After incubation, the non-hybridized nucleic acids areremoved, and the presence and amount of nucleic acids that havehybridized to the probes are detected and quantified.

Detection of nucleic acid molecules comprising polynucleotide sequencescoding for a protein marker can involve amplification of specificpolynucleotide sequences using an amplification method such as PCR,followed by analysis of the amplified molecules using techniques knownin the art. Suitable primers can be routinely designed by one skilled inthe art. In order to maximize hybridization under assay conditions,primers and probes employed in the methods of the invention generallyhave at least about 60%, preferably at least about 75% and morepreferably at least about 90% identity to a portion of nucleic acidsencoding a protein marker.

Hybridization and amplification techniques described herein can be usedto assay qualitative and quantitative aspects of expression of nucleicacid molecules comprising polynucleotide sequences coding for theinventive protein markers.

Alternatively, oligonucleotides or longer fragments derived from nucleicacids encoding each protein marker can be used as targets in amicroarray. A number of different array configurations and methods oftheir production are known to those skilled in the art (see, forexample, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974;5,384, 261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327;5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554, 501; 5,561,071;5,571,639; 5,593,839; 5,599,695; 5,624, 711; 5,658,734; and 5,700,637).Microarray technology allows for the measurement of the steady-statelevel of large numbers of polynucleotide sequences simultaneously.Microarrays currently in wide use include cDNA arrays andoligonucleotide arrays. Analyses using microarrays are generally basedon measurements of the intensity of the signal received from a labeledprobe used to detect a cDNA sequence from the sample that hybridizes toa nucleic acid probe immobilized at a known location on the microarray(see, for example, U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and6,271,002). Array-based gene expression methods are known in the art andhave been described in numerous scientific publications as well as inpatents (see, for example, M. Schena et al., Science, 1995, 270:467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93:10614-10619; 1. 1. Chen et al., Genomics, 1998, 51: 313324; U.S. Pat.Nos. 5,143,854; 5,445,934; 5,807,522; 5,837, 832; 6,040,138; 6,045,996;6,284,460; and 6,607,885).

Any biomarker binding agent, such as an antibody, can be labeled with aradiolabel or a fluorescent label. A labeled biomarker binding agent canbe administered into a subject, by any suitable method, such as byinjection. In some embodiments, a labeled biomarker binding agent can beadministered locally, such as to a chronic wounds such as pressureulcers, venous ulcers, stasis ulcers, venous stasis ulcers, diabeticfoot ulcers, arterial insufficiency ulcers or any combination thereof.The labeled biomarker binding agent can be detected by any suitablemeans known in the art. Exemplary instruments that can be used to detectradiolabeled agents or fluorescent agents after administration to asubject include, but are not limited to, instruments for IVIS Imaging™(Calipur), bioluminescence imaging (BLI), fluorescence-lifetime imaging(FLI) microscopy, X-ray radiography, ultrasound imaging, computedtomography (CT) imaging, single-photon emission computed tomography(SPECT), positron emission tomography (PET), magnetic resonance imaging(MRI), or any combination thereof. A labeled biomarker agent can bind toits respective biomarker upon administration of the agent into asubject. In some embodiments, the intensity of the signal from the labeland the region in a subject's body where the label accumulates canindicate a chronic wound such as pressure ulcers, venous ulcers, stasisulcers, venous stasis ulcers, diabetic foot ulcers, arterialinsufficiency ulcers or any combination thereof, where treatment isneeded.

Therapeutic Methods

Any method known in the art can be used to treat the chronic wound, orto treat the pathology that can be causing the chronic wound. A methodcan comprise treatment of a chronic wound in a mammal, such as aneuropathic ulcer decubitus ulcer, a venous ulcer or a diabetic ulcer oran infected wound. The method can comprise applying an A2M compositionto the wound. A2M compositions and formulations can be used forinhibiting proteases. A2M compositions can be used to prevent, slow, oralter FAC formation. A variant A2M can be more efficient than awild-type A2M polypeptide in inhibiting proteases, have a longerhalf-life, have a slower clearance factor, or any combination thereof.

In some embodiments, the wound is a decubital ulcer, a pressure ulcer, alower extremity ulcer, a deep sternal wound, a post-operative wound, arefractory post-operative wound of the trunk area, a wound to the greatsaphenous vein following harvesting of the great saphenous vein, avenous ulcer, or an anal fissure. In those embodiments involving a lowerextremity ulcer, the ulcer may be in a diabetic patient. In otherembodiments, the wound is a venous ulcer, pressure ulcer, orpost-operative ulcer.

The A2M composition can be comprised on a wound dressing. Dry andhydrated, i.e. wet wound dressings and delivery systems can be used andcan also be suitable for active ingredients, their use for the treatmentof wounds and skin diseases, preferably chronic wounds. A wound dressingcan be applied to the chronic wound for a period of at least 1 hour, atleast 24 hours, at least 48 hours, or at least 72 hours. The treatmentmay be extended for several days, weeks or months, with dressing changesas appropriate, if necessary for chronic wounds.

Another aspect of the invention relates to articles of manufacturecomprising a composition of the invention and a dressing. In someembodiments, the dressing is a dry dressing, moisture-keeping barrierdressing, or bioactive dressing. In those embodiments involving a drydressing, the dressing may be a gauze, a bandage, a non-adhesive mesh, amembrane, foils, foam, or a tissue adhesive. In those embodimentsinvolving a moisture-keeping barrier dressing, the dressing may be apaste, a cream, an ointment, a nonpermeable or semi-permeable membraneor foil, a hydrocolloid, a hydrogel, or combinations thereof. In thoseembodiments involving a bioactive dressing, the dressing may be anantimicrobial dressing. For example, the wound dressing may be a woven,nonwoven or knitted fabric having the A2M composition coated thereon, orit may be a bioresorbable polymer film or sponge having the A2Mcomposition dispersed therein for sustained release at the ulcer site.

Dressings and Matrices

In one aspect, one or more active agents are provided in the form of adressing or matrix. In certain embodiments, the one or more agents ofthe invention are provided in the form of a liquid, semi-solid or solidcomposition for application directly, or the composition is applied tothe surface of, or incorporated into, a solid contacting layer such as adressing gauze or matrix. The dressing composition may be provided forexample, in the form of a fluid or a gel. One or more active agents maybe provided in combination with conventional pharmaceutical excipientsfor topical application. Suitable carriers include: Pluronic gels,Poloxamer gels, Hydrogels containing cellulose derivatives, includinghydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethylcellulose, hydroxypropylmethyl cellulose and mixtures thereof; andhydrogels containing polyacrylic acid (Carbopols). Suitable carriersalso include creams/ointments used for topical pharmaceuticalpreparations, e.g., creams based on cetomacrogol emulsifying ointment.The above carriers may include alginate (as a thickener or stimulant),preservatives such as benzyl alcohol, buffers to control pH such asdisodium hydrogen phosphate/sodium dihydrogen phosphate, agents toadjust osmolarity such as sodium chloride, and stabilizers such as EDTA.

Suitable dressings or matrices may include, for example, the followingwith A2M compositions or formulations:

Suitable absorptives may include, for example, absorptive dressings,which can provide, for example, a semi-adherent quality or anon-adherent layer, combined with highly absorptive layers of fibers,such as for example, cellulose, cotton or rayon. Alternatively,absorptives may be used as a primary or secondary dressing.

Suitable alginates include, for example, dressings that are non-woven,non-adhesive pads and ribbons composed of natural polysaccharide fibersor xerogel derived from seaweed. Suitable alginates dressings may, forexample, form a moist gel through a process of ion exchange upon contactwith exudate. In certain embodiments, alginate dressings are designed tobe soft and conformable, easy to pack, tuck or apply overirregular-shaped areas. In certain embodiments, alginate dressings maybe used with a second dressing.

Suitable antimicrobial dressings may include, for example, dressingsthat can facilitate delivery of bioactive agents, such as, for example,silver and polyhexamethylene biguanide (PHMB), to maintain efficacyagainst infection, where this is needed or desirable. In certainembodiments, suitable antimicrobial dressings may be available as forexample, as sponges, impregnated woven gauzes, film dressings,absorptive products, island dressings, nylon fabric, non-adherentbarriers, or a combination of materials.

Suitable biological dressings or biosynthetic dressings may include, forexample, gels, solutions or semi-permeable sheets derived from a naturalsource, e.g., pigs or cows. In certain embodiments, a gel or solution isapplied to the treatment site and covered with a dressing for barrierprotection. In another embodiment, a biological-based orbiosynthetic-based sheet is placed in situ which may act as membrane,remaining in place after a single application, or the biologicaldressings or biosynthetic dressings may be prepared in advance toinclude the therapeutics agents.

Suitable collagen dressings may include, for example, gels, pads,particles, pastes, powders, sheets or solutions derived from forexample, bovine, porcine or avian sources or other natural sources ordonors. In certain embodiments, the collagen dressing may interact withtreatment site exudate to form a gel. In certain embodiments, collagendressing may be used in combination with a secondary dressing.

Suitable composite dressings may include, for example, dressings thatcombine physically distinct components into a single product to providemultiple functions, such as, for example, a bacterial barrier,absorption, and adhesion. In certain embodiments, the compositedressings are comprised of, for example, multiple layers and incorporatea semi- or non-adherent pad. In certain embodiments, the composite mayalso include for example, an adhesive border of non-woven fabric tape ortransparent film. In certain other embodiments, the composite dressingmay function as for example, either a primary or a secondary dressingand in yet another embodiment, the dressing may be used in combinationwith topical pharmaceutical composition.

Suitable contact layer dressings may include, for example, thin,non-adherent sheets placed on an area to protect tissue from forexample, direct contact with other agents or dressings applied to thetreatment site. In certain embodiments, contact layers may be deployedto conform to the shape of the area of the treatment site and are porousto allow exudate to pass through for absorption by an overlying,secondary dressing. In yet another embodiment, the contact layerdressing may be used in combination with topical pharmaceuticalcomposition.

Suitable elastic bandages may include, for example, dressings thatstretch and conform to the body contours. In certain embodiments, thefabric composition may include for example, cotton, polyester, rayon, ornylon. In certain other embodiments, the elastic bandage may forexample, provide absorption as a second layer or dressing, to hold acover in place, to apply pressure or to cushion a treatment site.

Suitable foam dressings may include, for example, sheets and othershapes of foamed polymer solutions (including polyurethane) with small,open cells capable of holding fluids. Exemplary foams may be forexample, impregnated or layered in combination with other materials. Incertain embodiments, the absorption capability may be adjusted based onthe thickness and composition of the foam. In certain other embodiments,the area in contact with the treatment site may be non-adhesive for easyremoval. In yet another embodiment, the foam may be used in combinationwith an adhesive border and/or a transparent film coating that can serveas an anti-infective barrier.

Suitable gauze dressings and woven dressings may include, for example,dry woven or non-woven sponges and wraps with varying degrees ofabsorbency. Exemplary fabric composition may include, for example,cotton, polyester, or rayon. In certain embodiments, gauzes andnon-woven dressing may be available sterile or non-sterile in bulk andwith or without an adhesive border. Exemplary gauze dressings and wovendressings may be used for cleansing, packing and covering a variety oftreatment sites.

Suitable hydrocolloid dressings may include, for example, wafers,powders or pastes composed of gelatin, pectin, orcarboxymethylcellulose. In certain embodiment, wafers are self-adheringand available with or without an adhesive border and in a wide varietyof shapes and sizes. Exemplary hydrocolloids are useful on areas thatrequire contouring. In certain embodiments, powders and pasteshydrocolloids may use used in combination with a secondary dressing.

Suitable amorphous hydrogel dressings may include, for example,formulations of water, polymers and other ingredients with no shape,designed to donate moisture and to maintain a moist healing environmentsand or to rehydrate the treatment site. In certain embodiments,hydrogels may be used in combination with a secondary dressing cover.Suitable impregnated hydrogel dressings may include, for example, gauzesand non-woven sponges, ropes and strips saturated with an amorphoushydrogel. Amorphous hydrogels may include for example, formulations ofwater, polymers and other ingredients with no shape, designed to donatemoisture to a dry treatment site and to maintain a moist healingenvironment.

Suitable hydrogel sheets may include for example, three-dimensionalnetworks of cross-linked hydrophilic polymers that are insoluble inwater and interact with aqueous solutions by swelling. Exemplaryhydrogels are highly conformable and permeable and can absorb varyingamounts of drainage, depending on their composition. In someembodiments, the hydrogel is non-adhesive against the treatment site ortreated for easy removal.

Suitable impregnated dressings may include, for example, gauzes andnon-woven sponges, ropes and strips saturated with a solution, anemulsion, oil, gel or some other pharmaceutically active compound orcarrier agent, including for example, saline, oil, zinc salts,petrolatum, xeroform, and scarlet red as well as the compounds describedherein. Silicone Gel Sheets: suitable silicone gel sheet dressings mayinclude, for example, soft covers composed of cross-linked polymersreinforced with or bonded to mesh or fabric.

Suitable liquid dressings may include, for example, mixtures ofmultiprotein material and other elements found in the extracellularmatrix. In certain embodiments, exemplary solutions may be applied tothe treatment site after debridement and cleansing and then covered withan absorbent dressing or a nonadherent pad. Transparent Films: suitabletransparent film dressings may include polymer membranes of varyingthickness coated on one side with an adhesive. In certain embodiments,transparent films are impermeable to liquid, water and bacteria butpermeable to moisture vapor and atmospheric gases. In certainembodiments, the transparency allows visualization of the treatmentsite.

Suitable filler dressings may include, for example, beads, creams,foams, gels, ointments, pads, pastes, pillows, powders, strands, orother formulations. In certain embodiments, fillers are non-adherent andmay include a time-released antimicrobial. Exemplary fillers may beuseful to maintain a moist environment, manage exudate, and fortreatment of for example, partial- and full-thickness wounds, infectedwounds, draining wounds, and deep wounds that require packing. Where theA2M composition is used for prophylaxis of chronic wounds, conventionaltransdermal pharmaceutical forms may be appropriate, such asslow-release skin patches to prevent or minimize ulcer formation orbreakout. More conventional systemic administration, such as oral orparenteral administration, may be preferable.

In still another aspect, a composition of the invention may furthercomprise as a matrix or scaffold a material suitable for implantation ina person. In some embodiments, the material is a solid beforeimplantation. In some embodiments, the material is a gel that solidifiesfollowing implantation.

The A2M compositions may be suitable for local or systemic, oral orparenteral administration. However, preferably, the composition is inthe form of an ointment for topical administration to a chronic wound orulcer. The ointment can comprise an A2M composition in apharmaceutically acceptable carrier. Suitable carriers include:Hydrogels containing cellulose derivatives, including hydroxyethylcellulose, hydroxymethyl cellulose, carboxymethyl cellulose,hydroxypropylmethyl cellulose and mixtures thereof; and hydrogelscontaining polyacrylic acid (Carbopols). Suitable carriers alsoincluding creams/ointments used for topical pharmaceutical preparations,e.g., creams based on cetomacrogol emulsifying ointment. The abovecarriers may include alginate (as a thickener or stimulant),preservatives such as benzyl alcohol, buffers to control pH such asdisodium hydrogen phosphate/sodium dihydrogen phosphate, agents toadjust osmolarity such as sodium chloride, and stabilizers such as EDTA.

Wound dressing compositions can be located on an inert support, such asan adhesive strip, adhesive wrap, bandage, gauze bandage or compresssystem. Wound dressing compositions, systems and packages can be usedfor the treatment of wounds, especially badly healing wounds likechronic wounds, in particular for the treatment of diabetic, venous,decubitus or neuropathic ulcers or infected wounds. For example, A2Mcompositions or formulation can be directly smeared 1 to 3 mm thick orthinner than 2 mm thick on the wound surface. The wound surface can becovered and/or bound, such as with cotton gauze or a ventilativematerial. The dressing can be removed one or more times during a timeperiod. For example, the dressing can be removed 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more times over a 12-96 hours. The dressing change can removemetabolites in wound surfaces. The wound can then be covered with an A2Mcomposition and bound with gauze dressing. Necrotic musculotendinoustissue can be removed. In some embodiments, disinfectants are notapplied to the surface of the wound.

Any number of methods known in the art for treating chronic wounds canbe applied to treat the patient. Suitable methods include surgical andnon-surgical methods including, but not limited to, arthroscopicdebridement or administration of steroidal or non-steroidalanti-inflammatory agents. Suitable methods include but are not limitedto, laminotomy, laminectomy, discectomy, microdiscectomy, percutaneousdiscectomy, endoscopic discectomy, laser discectomy, foramenotomy,fusion, prolotherapy, other surgical decompressions, decompression withfusion with or without instrumentation.

Chronic wounds can also be treated by standard non-surgical methods,including administration of steroidal or non-steroidal anti-inflammatoryagents. Non-steroidal anti-inflammatory (NSAID) agents are well known inthe art. Non-steroidal agents, including NSAIDs such as ibuprofen,aspirin or paracetamol can be used. Steroids, such as glucocorticoids,which reduce inflammation by binding to cortisol receptors, can also beused for treatment.

Other wound healing therapeutic substances can be used, such asnon-steroidal anti-inflammatory drugs, e.g., acetaminophen, steroids,like hydrocortisone or betamethosone, local anesthetics, antimicrobialagents, growth factors (e.g., fibroblast growth factors or plateletderived growth factor), or protease inhibitors. The antimicrobial agentmay, for example, comprise an antiseptic, an antibiotic, or mixturesthereof. Preferred antibiotics include cephalosporins (cephalexin,cefoxytin, and others), penicillins (amoxycillin, ampicillin,phenoxymethylpenicillin, and others), tetracyclines (minocycline,doxycycline, and others), aminoglycosides (gentamicin, neomycin, andothers), antifungals (isoconazole, clotrimazole, amphotericin, andothers), sulphadiazine, chloramphenicol, erythromycin, vancomycin,trimethoprim, and others. Preferred antiseptics include silver,including colloidal silver, silver salts including one or more silversalts of one or more of the anionic polymers making up the material,silver sulfadiazine, chlorhexidine, povidone iodine, triclosan,sucralfate, quarternary ammonium salts and mixtures thereof.

A2M compositions and formulations can be used for enhancing thenonspecific inhibition of one or more proteases in a human or non-humananimal experiencing or susceptible to one or more conditions selectedfrom the group of chronic wounds such as pressure ulcers, venous ulcers,stasis ulcers, venous stasis ulcers, diabetic foot ulcers, arterialinsufficiency ulcers or any combination thereof. For example, A2Mcompositions can be administered to an animal to reduce one or moreprotease activities in an animal.

Any of the compositions or formulations can be for administration byparenteral (intramuscular, intraperitoneal, intravenous (IV) orsubcutaneous injection), transdermal (either passively or usingiontophoresis or electroporation), or transmucosal (nasal, vaginal,rectal, or sublingual), oral, intra-articular or inhalation routes ofadministration. A2M compositions and formulations can also beadministered using bioerodible inserts, bare-metal stents (BMS), ordrug-eluting stents (DES or coated stents, or medicated stents), and canbe delivered directly to chronic wounds such as pressure ulcers, venousulcers, stasis ulcers, venous stasis ulcers, diabetic foot ulcers,arterial insufficiency ulcers or any combination thereof. A2Mcompositions and formulations can be formulated in dosage formsappropriate for each route of administration. An A2M containing an agentthat is not peptides or polypeptides, can additionally be formulated forenteral administration.

In some embodiments, the A2M compositions can be delivered by injection(peripherally or directly to a site). In one aspect, the injection ismade at or adjacent to a site or wound, e.g., 1-10 mm from the site orwound edge. In other embodiments, the injection is made about 1-8, 1-7,1-6, 1-5, 1-4, 1-3 and 1-2 mm from the site or wound edge. In stillother embodiments, the injection is made about 2-8, 2-7, 2-6, 2-5, 2-4and 2-3 mm from the site or wound edge. In one embodiment, the injectionis made 2-4 or 2-5 mm from the site or wound edge. In sites ofadministration, including wounds, which have length greater than about 1cm, the injections can occur once every linear centimeter. In oneembodiment, the injection is angled in toward a wound or other site ofadministration, or the injection is made into the dermis of a wound, orby intradermal, intra-tissue or intra-organ injection.

The A2M compositions and formulations can be administered to a subjectin a therapeutically effective amount. The precise dosage will varyaccording to a variety of factors such as subject dependent variables,such as age, the injury or pathology being treated, and the treatmentbeing affected. The exact dosage can be chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors that can betaken into account include the severity of the disease, age of theorganism, and weight or size of the organism; diet, time and frequencyof administration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Short acting pharmaceutical compositionsare administered daily whereas long acting pharmaceutical compositionsare administered every 2, 3 to 4 days, every week, or once every twoweeks. Depending on half-life and clearance rate of the particularformulation, the pharmaceutical compositions of the invention areadministered once, twice, three, four, five, six, seven, eight, nine,ten or more times per day.

For some compositions, the selected dosage depends upon the route ofadministration, and on the duration of the treatment desired. Generallydosage levels can include 0.1 to 40 mg/kg of body weight daily.Generally, for local injection or infusion, dosages can be lower.Depending on the composition and site of administration, dosage levelscan be between about 1 to 500,000 mg, in a volume between about 0.1 to10 mL. For example, dosage levels can be between about 5 to 450 mg, 5 to400 mg, 5 to 350 mg, 5 to 300 mg, 5 to 250 mg, 5 to 200 mg, 5 to 150 mg,5 to 100 mg, 5 to 500 mg, 5 to 25 mg, 100 to 150 mg, 100 to 200 mg, 100to 250 mg, 100 to 300 mg, 100 to 350 mg, 100 to 400 mg, 100 to 450 mg,or 100 to 500 mg in a volume between about 0.1 to 9 mL, 0.1 to 8 mL, 0.1to 7 mL, 0.1 to 6 mL, 0.1 to 5 mL, 0.1 to 4 mL, 0.1 to 3 mL, 0.1 to 2mL, 0.1 to 1 mL, 0.1 to 0.9 mL, 0.1 to 0.7 mL, 0.1 to 0.6 mL, 0.1 to 0.5mL, 0.1 to 0.4 mL, 0.1 to 0.3 mL, 0.1 to 0.2 mL, 1 to 9 mL, 1 to 8 mL, 1to 7 mL, 1 to 6 mL, 1 to 5 mL, 1 to 4 mL, 1 to 3 mL, or 1 to 2 mL.Normal dosage amounts of various variant A2M polypeptides or nucleicacids, or fragment thereof can vary from any number betweenapproximately 1 to 500,000 micrograms, up to a total dose of about 50grams, depending upon the route of administration. Desirable dosagesinclude, for example, 250 μg, 500 μg, 1 mg, 50 mg, 100 mg, 150 mg, 200mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g,1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 3 g, 4 g, 5, 6 g, 7 g, 8g, 9 g, 10 g, 20 g, 30 g, 40 g, and 50 g.

The dose of the composition can be administered to produce a tissue orblood concentration from approximately any number between 0.1 μM to 500mM. Desirable doses produce a tissue or blood concentration of about anynumber from 1 to 800 μM. Preferable doses produce a tissue or bloodconcentration of greater than about any number from 10 μM to about 500μM. For example, a dose can comprise the amount of active ingredientrequired to achieve a tissue or blood concentration of 10 μM, 15 μM, 20μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 110 μM, 120 μM, 130 μM,140 μM, 145 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 220 μM,240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM,400 μM, 420 μM, 440 μM, 460 μM, 480 μM, and 500 μM. Although doses thatproduce a tissue concentration of greater than 800 μM are not preferred,they can be used with some embodiments of the invention. A constantinfusion can also be provided so as to maintain a stable concentrationin the tissues as measured by blood levels.

Any of the A2M compositions or formulations can be administered in anaqueous solution by parenteral, intradiscal, intrafacet, intrathecal,epidural or topical application. Any composition described herein can beadministered directly to the chronic wound. For example, whenfibronectin-aggrecan complexes are detected in the wound, an A2Mcomposition can be administered by direct injection into the wound.Alternatively, the compositions can be administered by directapplication to the wound when FACs or protease activity or othersuitable biomarkers are detected in these spaces. For example, aggrecancan include any naturally-occurring variants and splice variants ofaggrecan, versican, brevican and neurocan, and any variants of aggrecan,versican, brevican and neurocan due to splicing by different cell types.Fibronectin can include any naturally occurring fibronectin variantsincluding approximately 20 known splice variants associated with adisease or a disorder and fibronectin variants due to different splicingby different cell types.

A composition or formulation can also be in the form of a suspension oremulsion. In general, pharmaceutical compositions are provided includingeffective amounts of a peptide or polypeptide, and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents sterile water, buffered saline of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally,additives such as detergents and solubilizing agents (e.g., TWEEN® 20,TWEEN® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations can be lyophilizedand redissolved or resuspended immediately before use. The formulationcan be sterilized by, for example, filtration through a bacteriaretaining filter, by incorporating sterilizing agents into thecompositions, by irradiating the compositions, or by heating thecompositions. In some embodiments, linoleic acid can be used in the A2Mcompositions, for example, to treat a diabetic ulcer.

Any composition or formulation can also be administered in controlledrelease formulations. Controlled release polymeric devices can be madefor long term release systemically following implantation of a polymericdevice (rod, cylinder, film, or disc) or injection (microparticles). Thematrix can be in the form of microparticles such as microspheres, wherepeptides are dispersed within a solid polymeric matrix or microcapsules,where the core can be of a different material than the polymeric shell,and the peptide can be dispersed or suspended in the core, which can beliquid or solid in nature. Unless specifically defined herein,microparticles, microspheres, and microcapsules are usedinterchangeably. Alternatively, the polymer can be cast as a thin slabor film, ranging from nanometers to four centimeters, a powder producedby grinding or other standard techniques, or even a gel such as ahydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of any composition or formulation described herein, althoughbiodegradable matrices are preferred. These can be natural or syntheticpolymers, although synthetic polymers are preferred due to the bettercharacterization of degradation and release profiles. The polymer can beselected based on the period over which release can be desired. In somecases linear release can be most useful, although in others a pulserelease or “bulk release” can provide more effective results. Thepolymer can be in the form of a hydro gel (typically in absorbing up toabout 90% by weight of water), and can optionally be crosslinked withmultivalent ions or polymers. The matrices can be formed by solventevaporation, spray drying, solvent extraction and other methods known tothose skilled in the art. Bioerodible microspheres can be prepared usingany of the methods developed for making microspheres for drug delivery,for example, as described by Mathiowitz and Langer, J. ControlledRelease, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers,6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci.,35:755-774 (1988).

Devices can be formulated for local release to treat the area ofimplantation or injection which will typically deliver a dosage that canbe much less than the dosage for treatment of an entire body or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

Any A2M composition or formulation can be used in the treatment of acondition or a disease. For example, a condition or disease can bechronic wounds such as pressure ulcers, venous ulcers, stasis ulcers,venous stasis ulcers, diabetic foot ulcers, arterial insufficiencyulcers or any combination thereof.

In some embodiments, agents that are not peptides or polypeptides, canbe used in the treatment of a condition or a disease. For example, anagent, can be administered directly onto a chronic wound such aspressure ulcers, venous ulcers, stasis ulcers, venous stasis ulcers,diabetic foot ulcers, arterial insufficiency ulcers or any combinationthereof.

Any of the compositions described herein can be isolated from a bloodsample and can be suitable for administration to chronic wounds such aspressure ulcers, venous ulcers, stasis ulcers, venous stasis ulcers,diabetic foot ulcers, or arterial insufficiency ulcers.

In other aspects, the invention is directed to a method of inhibitingthe onset of infection in a wound, comprising administering to the wounda composition of the invention. In one embodiment, the wound is causedby trauma. In another embodiment, the wound is caused by surgery.

In those aspects involving methods of treating a wound, the wound mayalso be treated by administering to the wound an article of manufacturecomprising a composition of the invention.

For purposes of the present invention, wounds include tattoos.Accordingly, a further aspect of the present invention is directed to amethod of tattoo removal, which includes administering or otherwiseapplying the A2M composition or an article of manufacture comprising theA2M composition, to a tattoo.

A wound dressing can be packaged in a microorganism-impermeablecontainer. Such packages can represent single or multi-unit dosage formsof the present compositions. These packages can be used by the subjecthimself.

The A2M composition may be used in conjunction with any otherconventional wound treatment, such as negative pressure, warming(therapeutic heat), electrical stimulation, magnetism, laserphototherapy, cycloidal vibration therapy and ultrasound. It also can beused with biological therapy such as larva therapy, skin substitutes,cultured keratinocytes (Epicel, Genzyme biosurgery), human dermalreplacement (Dermagraft, Smith and Nephew Inc.), cadaver derivedprocessed dermis (Alloderm, Life Cell Corporation), Bilayered SkinEquivalent (Apligraf, Organogenesis Inc.), TransCyte (Smith and NephewInc.), Growth Factors (PDGF is currently the only growth factor licensedfor topical use), and fibrin sealant. In some embodiments, the A2Mcomposition is used in conjunction with negative pressure wound therapy(NPWT) (one example being the V.A.C., which is a commercially availablewound therapy manufactured by KCI). Negative pressure therapy promoteswound healing by applying negative pressure to a wound. In theseembodiments, A2M composition can be applied to a wound prior to negativepressure therapy. In yet other embodiments, the A2M composition is usedin conjunction with hyperbaric oxygen therapy (Thackham, 2008) or ozonetherapy. For example, the A2M composition can be applied to a wound justprior to a patient receiving hyperbaric therapy. The A2M composition mayalso be used in conjunction with low-energy shock wave therapy (e.g.,impulses of about 0.1 mJ/mm2; 5 Hz) per centimeter of wound length).See, e.g., Dumfarth, et al., Ann Thorac. Surg. 86:1909-13 (2008).

Subjects

Any subject in need of treatment for a condition or disease describedherein, such as a subject with a chronic wound, can be treated with anycomposition or formulation described herein.

As used herein, a “condition” or “disease” is any disorder, disease, orcondition that would benefit from an agent that initiates, accelerates,promotes or enhances wound healing (including acute wounds, dehiscentwounds, and slow-healing delayed-healing and chronic wounds), reducesinflammation, reduces or lessens scarring, improves scar quality,reduces fibrosis, and/or reduces adhesions. For example, diseases,disorders, and conditions include acute wounds. Diseases, disorders, andconditions also include dehiscent wounds, and slow-healingdelayed-healing and chronic wounds. Also included are diseases,disorders, and conditions characterized by excess production of fibrousmaterial, including excess production of fibrous material within theextracellular matrix. Also included are diseases, disorders andconditions characterized by replacement of normal tissue elements byabnormal, non-functional, and/or excessive accumulation ofmatrix-associated components. Also included are diseases, disorders andconditions characterized by adhesion formation. Also included is anydisorder, disease, or condition that would benefit from an agent thatpromotes wound healing and/or reduces swelling, inflammation, and/orscar formation (including abnormal and excessive scarring, includingkeloid scars, hypertrophic scars, widespread (stretched) scars, andatrophic (depressed) scars). For example, included are wounds resultingfrom surgery or trauma, wounds that do not heal at expected rates (suchas delayed-healing wounds, incompletely healing wounds, chronic wounds,and dehiscent wounds), and wounds associated abnormalities in connectionwith neuropathic, ischemic, microvascular pathology, pressure over bonyarea (tailbone (sacral), hip (trochanteric), buttocks (ischial), or heelof the foot), reperfusion injury, and valve reflux etiology andconditions. Also included are diseases, disorders and conditions thatwould benefit from enhanced cellular migration, lessened cellularadhesion, scarring and inflammation as described herein. In someembodiments, a subject can be diagnosed with a condition or diseasebefore or after being diagnosed with a condition or disease, such as bythe methods described in U.S. Pat. No. 7,709,215 and U.S. PublicationNo.: US 2010/0098684A1. In some embodiments, a subject can be diagnosedas needing treatment with any of the compositions or formulationsdescribed herein.

Subjects can include any subject that presents with a chronic wound suchas pressure ulcers, venous ulcers, stasis ulcers, venous stasis ulcers,diabetic foot ulcers, arterial insufficiency ulcers or any combinationthereof. In some embodiments, a subject can be selected for thedetection of A2M. Subjects can have chronic wounds such as pressureulcers, venous ulcers, stasis ulcers, venous stasis ulcers, diabeticfoot ulcers, arterial insufficiency ulcers or any combination thereof.

At the time of treatment, a subject may have been experiencing chronicwounds for 30 or 25 weeks or less. For example, a subject may have beenexperiencing chronic wounds for 20, 15, 10, 8, or 6 weeks, or less.

Subjects can be of either sex and can be of any age.

A subject can be human or non-human animal. A subject can be a mammal,such as a mouse, rat, rabbit, cat, dog, monkey, horse or goat.Preferably the subject is human. A subject can be a non-human primate,rodent, caprine, bovine, ovine, equine, canine, feline, mouse, rat,rabbit, horse or goat.

Samples

Any of the autologous compositions described herein can be derived froma biological sample. Preferably, the autologous compositions describedherein are isolated from a biological sample and suitable for deliveryinto or onto a chronic wound. Biological samples include blood, sectionsof tissues such as biopsy samples, frozen sections taken for histologicpurposes, and lavage samples.

A biological sample can be a tissue sample or bodily fluid, such as ahuman bodily fluid. For example, the bodily fluid can be blood, sera,plasma, lavage, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen, prostatic fluid, Cowper'sfluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cystfluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme,chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretion,mucosal secretion, stool water, pancreatic juice, lavage fluid fromsinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, fluidfrom a chronic wound or umbilical cord blood. One or more of thebiological sample(s) can comprise a cell, such as a stem cell,undifferentiated cell, differentiated cell, or cell from a diseasedsubject or subject with a specific condition. A biological sample can beblood, a cell, a population of cells, a quantity of tissue, fluid, or asample from a chronic wound. A biological sample can comprise cells fromcartilaginous tissue or can be free of cells. A biological sample can besubstantially depleted of a common serum protein, such as, but notlimited to, albumin or IgG. Depletion can comprise filtration,fractionation, or affinity purification. A biological sample can be fromanimal, such as a mammal, for example, a human, non-human primate,rodent, caprine, bovine, ovine, equine, canine, feline, mouse, rat,rabbit, horse or goat

Pooled Samples

In some embodiments, pooled samples can be used to treat a disease orcondition, such as a wound. In some embodiments, two or more samples canbe obtained from two or more sources, such as two or more subjects, andpooled together before or after being processed into the A2Mconcentrated compositions described herein. However, such pooled samplesshould not be immunogenic to the subject being treated. For example, afirst blood sample can be obtained from a first subject and a secondblood sample can be obtained from a second subject. The blood samplescan be processed using a system containing two or more filters asdescribed herein. The processed samples can be substantially free ofblood cells. These samples can be pooled together and can be used totreat the first subject, the second subject, or a third subject, as longas the pooled sample is not immunogenic to the subject being treated.

A sample for the preparation of an autologous composition can be from asingle subject. For example, a blood sample taken from a single subjectcan be used to prepare an autologous sample described herein. A samplecan also include multiple sample taken from the same individual. Forexample, two or more blood samples can be obtained from a subject, andeach of the two or more samples taken from the subject can be used toprepare 2 or more autologous samples containing a concentrated amount ofA2M. These samples can be pooled together and used to treat the subjectfrom whom they were obtained.

Samples can also be obtained from two or more subjects, pooled together,and used to treat one of the subjects from which they were obtained. Thesamples can be pooled prior to or after being processed into acomposition containing concentrated A2M, such as using a method orsystem described herein. For example, blood from two or more subjectscan be pooled, or concentrated A2M samples obtained from two or moredifferent people can be pooled. The pooled samples can then be used totreat one of the subjects from which they were obtained, so long as thepooled sample is not immunogenic to the subject being treated with thepooled samples.

Samples can also be obtained from two or more subjects, pooled together,and used to treat a different subject than the subjects from whom theywere obtained. The samples can be pooled prior to or after beingprocessed into a composition containing concentrated A2M, such as usinga method or system described herein. For example, blood from two or moresubjects can be pooled, or concentrated A2M samples obtained from two ormore different people can be pooled. The pooled samples can then be usedto treat a different subject than the subjects from which they wereobtained, so long as the pooled sample is not immunogenic to the subjectbeing treated with the pooled samples.

Biological samples can be collected by any non-invasive means, such as,for example, by drawing blood from a subject, or using fine needleaspiration, swabbing a wound, or taking a sample from a wound, or needlebiopsy. Alternatively, biological samples can be collected by aninvasive method, including, for example, surgical biopsy.

A biological sample can comprise disease or condition specific proteins.A biological sample can be from a subject with a disease or condition orfrom a subject without a disease or condition. In some embodiments, abiological sample can be from a subject diagnosed with a disease orcondition or from a subject not diagnosed with or without a disease orcondition. A diagnosis can be made by any of the methods describedherein. A biological sample can be from a subject at one time point andanother biological sample can be from a subject at a later or earliertime point, wherein the subject can be the same or a different subject.For example, the subject may have a disease or condition or have beendiagnosed with a disease or condition, and samples can be taken as thedisease or condition progresses. A biological sample can be from asubject pretreatment and another biological sample can be from a subjectat post treatment, wherein the subject can be the same or differentsubject. A biological sample can be from a subject non-responsive totreatment and another biological sample can be from a subject responsiveto a treatment. Biological samples can be from the same or differentspecies. One or more biological samples can be from the same subject orfrom a different subject from which one or more other biological sampleswere obtained.

The methods of the invention can be applied to the study of any type ofbiological samples allowing one or more biomarkers to be assayed. Abiological sample can be a fresh or frozen sample collected from asubject, or archival samples with known diagnosis, treatment and/oroutcome history.

The inventive methods can be performed on the biological sample itselfwithout or with limited processing of the sample. The inventive methodscan be performed at the single cell level (e.g., isolation of cells fromthe biological sample). Multiple biological samples can be taken fromthe same tissue/body part in order to obtain a representative samplingof the tissue.

Any of the method described herein can be performed on a protein extractprepared from the biological sample. The methods can also be performedon extracts containing one or more of: membrane proteins, nuclearproteins, and cytosolic proteins. Methods of protein extraction are wellknown in the art (see, for example “Protein Methods”, D. M. Bollag etal., 2nd Ed., 1996, Wiley-Liss; “Protein Purification Methods: APractical Approach”, E. L. Harris and S. Angal (Eds.), 1989; “ProteinPurification Techniques: A Practical Approach”, S. Roe, 2nd Ed., 2001,Oxford University Press; “Principles and Reactions o/Protein Extraction,Purification, and Characterization”, H. Ahmed, 2005, CRC Press: BocaRaton, Fla.). Numerous different and versatile kits can be used toextract proteins from bodily fluids and tissues, and are commerciallyavailable from, for example, BioRad Laboratories (Hercules, Calif.), BDBiosciences Clontech (Mountain View, Calif.), Chemicon International,Inc. (Temecula, Calif.), Calbiochem (San Diego, Calif.), PierceBiotechnology (Rockford, Ill.), and Invitrogen Corp. (Carlsbad, Calif.).After the protein extract has been obtained, the protein concentrationof the extract can be standardized to a value being the same as that ofthe control sample in order to allow signals of the protein markers tobe quantitated. Such standardization can be made using photometric orspectrometric methods or gel electrophoresis.

Any of the method described herein can be performed on nucleic acidmolecules extracted from the biological sample. For example, RNA can beextracted from the sample before analysis. Methods of RNA extraction arewell known in the art (see, for example, J. Sambrook et al., “MolecularCloning: A Laboratory Manual”, 1989, 2nd Ed., Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.). Most methods of RNAisolation from bodily fluids or tissues are based on the disruption ofthe tissue in the presence of protein denaturants to quickly andeffectively inactivate RNAses. Isolated total RNA can then be furtherpurified from the protein contaminants and concentrated by selectiveethanol precipitations, phenol/chloroform extractions followed byisopropanol precipitation or cesium chloride, lithium chloride or cesiumtrifluoroacetate gradient centrifugations. Kits are also available toextract RNA (i.e., total RNA or mRNA) from bodily fluids or tissues andare commercially available from, for example, Ambion, Inc. (Austin,Tex.), Amersham Biosciences (Piscataway, N.J.), BD Biosciences Clontech(Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.), GIBCO BRL(Gaithersburg, Md.), and Qiagen, Inc. (Valencia, Calif.).

After extraction, mRNA can be amplified, and transcribed into cDNA,which can then serve as template for multiple rounds of transcription bythe appropriate RNA polymerase. Amplification methods are well known inthe art (see, for example, A. R. Kimmel and S. L. Berger, MethodsEnzymol. 1987, 152: 307-316; J. Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 1989, 2nd Ed., Cold Spring Harbour Laboratory Press:New York; “Short Protocols in Molecular Biology”, F. M. Ausubel (Ed.),2002, 5th Ed., John Wiley & Sons; U.S. Pat. Nos. 4,683,195; 4,683,202and 4,800,159). Reverse transcription reactions can be carried out usingnon-specific primers, such as an anchored oligo-dT primer, or randomsequence primers, or using a target-specific primer complementary to theRNA for each probe being monitored, or using thermostable DNApolymerases (such as avian myeloblastosis virus reverse transcriptase orMoloney murine leukemia virus reverse transcriptase).

Other Embodiments

Disclosed herein are molecules, materials, compositions, and componentsthat can be used for, can be used in conjunction with, can be used inpreparation for, or are products of methods and compositions disclosedherein. It is understood that when combinations, subsets, interactions,groups, etc. of these materials are disclosed and while specificreference of each various individual and collective combinations andpermutation of these molecules and compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a nucleotide or nucleic acid is disclosed and discussed anda number of modifications that can be made to a number of moleculesincluding the nucleotide or nucleic acid are discussed, each and everycombination and permutation of nucleotide or nucleic acid and themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed molecules and compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

Those skilled in the art can recognize, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagents describedas these can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present disclosure which canbe limited only by the appended claims.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention that are obvious tothose skilled in the art are intended to be within the scope of theinvention. Other embodiments are in the claims.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The followingreferences contain embodiments of the methods and compositions that canbe used herein: The Merck Manual of Diagnosis and Therapy, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007(ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia ofMol. Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Mol. Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Standard procedures of the present disclosure are described, e.g., inManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrooket al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl (eds.),Academic Press Inc., San Diego, USA (1987)). Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols inImmunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manualof Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), and Animal Cell Culture Methods (Methods in CellBiology, Vol. 57, Jennie P. Mather and David Barnes editors, AcademicPress, 1st edition, 1998), which are all incorporated by referenceherein in their entireties.

It should be understood that the following examples should not beconstrued as being limiting to the particular methodology, protocols,and compositions, etc., described herein and, as such, can vary. Thefollowing terms used herein are for the purpose of describing particularembodiments only, and are not intended to limit the scope of theembodiments disclosed herein.

EXAMPLES Example 1

A study was conducted to define the cell composition andAlpha-2-Macroglobulin (A2M) concentrations of concentrated plasmaproducts using different methods of production. Concentrated productswere produced by

-   -   1. pre-mixing RBC-reduced (RBC-R) PRP and PPP before filter        concentration,    -   2. post-mixing RBC-reduced PRP with filter concentrated PPP, or    -   3. adapting a centrifuge for an Autologous Platelet Integrated        Concentrate (APIC) 50 ml conical and filter-concentrating the        PRP obtained.

Concentration was performed with each method using a HemacorPolysulphone Hemaconcentrator Junior (APIC-HL HPH-Jr) or APIC-HH filter.The platelet, red blood cell, and white blood cells were counted in eachproduct and their intermediates. A2M concentration was obtained byEnzyme-Linked Immunosorbent Assay (ELISA) for each product as well.

From the study, it was concluded that:

-   -   1. Concentrated products made with the APIC-HH filter had higher        concentrations of platelets and A2M than in the HPH-Jr in each        method.    -   2. The highest A2M concentration achieved was by pre-mixing        RBC-R and PPP then concentrating on the APIC-HH filter (˜3.5×A2M        over PPP, ˜7×A2M over WB). The HPH-Jr filter had approximately a        2× drop in A2M concentration with the same protocol (˜2.5×A2M        over PPP, ˜5×A2M over WB)    -   3. Post-mixing the RBC-R and concentrated PPP resulted in        significantly reduced levels of platelet and A2M, (1-2.5× less        than pre-mixing).    -   4. Centrifuging a 50 ml conical within the parameters of a        centrifuge resulted in an ˜18 min spin time as well as much        increased WBC concentrations (1-1.5× of whole blood) compared to        their RBCR counterparts.

Example 2 Protocol

1. Autologous Blood Harvest

-   -   a. Prepare a 60 ml syringes containing 7.5 ml sterile ACDA.    -   b. Perform venipuncture and autologous blood harvest into        syringes.    -   c. Draw blood until a total volume of 50 ml is achieved. (42.5        ml blood+7.5 ml ACDA=48 ml Vf, 15% ACDA)        -   i. Transfer 5 ml of WB+ACDA to a 10 ml falcon tube using a            blunt-ended canula for later analysis.

2. Production of Plasma

-   -   a. Transfer the 45 ml whole blood+ACDA to a centrifuge tube        using a blunt-ended canula. Careful that the centrifuge is        balanced before spinning—you will need another centrifuge tube        full of 47.7 ml water. The specific density of blood is ˜1.06.    -   b. Centrifuge blood with the “PRP” setting (4 minutes, 1280×g,        brake 3).    -   c. Carefully remove the centrifuge tubes and place vertically in        the tube holder.    -   d. Attach a spinal needle to a 20 ml syringe and slowly draw        17.5 ml of plasma from about 1 cm above the buffy coat. Be        careful not to draw up any of the buffy coat into the syringe.        This will be Syringe #1 in FIGS. 4-6.        -   i. Transfer 2.5 ml of plasma to a 10 ml falcon tube for            later analysis.

3. Prime the APIC-PRP System

-   -   a. Adjust stopcock #1 so that the off position blocks access to        the unused port.    -   b. Adjust stopcock #2 to prevent access to Syringe #2.    -   c. All syringes should be in the completely compressed state to        prevent additional unwanted air in the system.    -   d. Tap Syringe #1 with Plasma to get all air bubbles to the top.        Remove the cap.    -   e. Holding the APIC-filter setup at an angle with Syringe #3 at        the top, attach Syringe #1 with Plasma to the APIC-PRP filter.    -   f. Slowly push the Plasma from Syringe #1 into the filter until        the sterile air from the filter and a few milliliters of plasma        are in Syringe #3. The filter should be devoid of air now.        Compress Syringe #3 until the plasma has just returned to        stopcock #2.    -   g. Rotate stopcock #2 to block Syringe #3 (sterile air) and        expose Syringe #2.    -   h. Pull back the plunger of Syringe #4 slightly to create a        slight space for filtrate to enter.    -   i. Attach the APIC Kit to the Styrofoam block to allow easy        pumping action.

4. Production of APIC-PRP

-   -   j. Sequentially compress Syringes #1 and #2 to process Plasma        over the filter. Filtrate will soon fill the inner filtrate        chamber of the filter and migrate into Syringe #4.        -   i. Monitor Syringe #4 for progress. If the plunger does not            migrate during processing a slight pull outwards will help            it get started.    -   k. If air bubbles are still in the system during processing,        tilt the system so they migrate into Syringe #2. During        processing, do not compress Syringe #2 completely to avoid        injecting air bubbles into the filter. It is much easier to        remove large air bubbles later than micro-bubbles caused by        processing bubbles through a filter.    -   l. As needed, unlock Syringe #4 and pull back slightly to create        room for additional filtrate.    -   m. Continue processing plasma into APIC until the desired amount        of concentration is achieved.        -   i. System void-volume is ˜2 ml.        -   ii. For a 4× volume concentration, 15 ml Plasma-PRP can be            processed until ˜2 ml are in Syringe #1.    -   n. Once the appropriate amount of concentration has been        achieved, compress Syringe #2 until the APIC has been        transferred to Syringe #1.    -   o. To remove excess APIC-PRP from the filter, turn the stopcock        to block Syringe #2 and allow access to Syringe #3 containing        the sterile air. Slowly compress Syringe #3 to push the APIC-PRP        in the filter into Syringe #1. Avoid adding air bubbles to        Syringe #1.    -   p. Syringe #1 containing APIC-PRP can be removed. Tap the        syringe to displace any air bubbles to the top for removal. If        any micro air bubbles are observed, place the Syringe with APIC        upright for 15 minutes until bubbles rise, remove air from        syringe. APIC is ready to use.

Example 3 Blood Collection

Autologous blood harvest was performed by venipuncture according topresent IRBs from healthy donors who were at least 18 years of age orolder, healthy, over 110 lbs, and not pregnant. Three Patients donated216 ml of whole blood mixed with 24 ml of Acid Citrate Dextrose SolutionA (ACDA) anticoagulant (10%) for the preparation of APIC-2 PRP and PPPin conditions #1 and #2, and 76 ml of whole blood mixed with 14 ml ofACDA (15%) for #3, for a total blood donation of 292 ml anticoagulatedblood. The anticoagulated blood was used as needed for the followingexamples.

Example 4 Preparation of APIC-2 PRP

Briefly, 60 ml of anticoagulated whole blood mixed with 10% ACDA wasadded to a first port in a vessel. Two ml of ACDA was further added to asecond port. The vessel was tapped to disperse blood to the bottom ofthe vessel. The vessel was then placed into a centrifuge and thecentrifuge started and allowed to complete the full cycle (14 min) A 30ml syringe with spacers was used to pull off the PPP contained in asmaller extension vessel by way of the white port. The PPP was saved forlater concentration. The PRP was mixed and collected by the 20 mlsyringe without spacers. The final ACDA concentration in the PRP and PPPwas approximately 15%, depending on the total volume of blood tipped.The amount of analytes in this sample were measured and compared to themeasurements of the same analytes in whole blood, plasma, and APIC-PRP;the concentration of cells platelets, WBC, and RBC (% of WB); theconcentration of A2M; and the concentration of PDGF ββ, PDGF αβ/ββ,bFGF, VEGF, and TGF-β1.

Example 5 Red Blood Cell-Reduced PRP (RBC-R)

The PRP from Example 3 was further processed to reduce the amount of redand white blood cells by centrifugation in a 10 ml tube using theadapter to the current Harvest centrifuge rotor. The PRP was placed inthis tube and centrifuged through the first part of the centrifugationcycle. The centrifuge was stopped manually as it completed braking, butbefore continuing to the second part of the cycle. The RBCR-PRP wasremoved by syringe to ˜1 cm above the buffy coat, leaving the packed RBCand WBC in the tube.

Example 6 50 ml Conical Spin

90 ml of whole blood mixed with 14 ml of ACDA was added to the 45 mlmark on two 50 ml tubes. The tubes were centrifuged in the Harvestcentrifuge using a separate rotor with buckets for 50 ml tubes. Thebuckets were taped to their swinging out positions to allow for the lidto close and brake without disturbing the tubes. The tubes werecentrifuged for 1.5 cycles (the 14 min complete cycle, then restartedfor the first part of another cycle) and manually stopped as the firstpart of the cycle came to a stop but before the second part of the cycleresumed. 15 ml of PRP was taken 1 cm from the buffy coat on each tubeand mixed together.

Example 7 Concentration of Plasma by APIC-HL (HPH-Jr) or APIC-HH Filters

The APIC-HL manual concentration was assembled and the void volume ofthe filter was measured by how much material went into the filter beforecoming out the other side (12 ml). The APIC-HH Filter was assembled,with a void volume of 2 ml.

Plasma was concentrated using the following method:

Holding the filters vertically, plasma was slowly injected from thebottom syringe (30 ml syringe) into the filter. Sterile air from thefilter was directed into the side 10 ml syringe. Once the filters hadbeen primed with plasma, stop-cocks were adjusted to permit plasma frombetween the 20 ml and 30 ml side syringes. Sequential compressions ofthe side syringes caused flow of plasma through the hollow fibermembranes. Filtrate accumulated in the 20 ml syringe attached to thefilter, whereas retentate remained in the side syringes. Oncecompressions were finished, the filters were held vertically and thestopcock adjusted to allow the sterile air back into the filter. The 10ml syringe was then compressed to void more of the product into thebottom syringe. Plasma was concentrated by the APIC-HL until there wasnothing left in the syringes to pump. The final amount of concentratedproduct was returned to the starting syringe by voiding the air from the10 ml syringe. Plasma was concentrated by the APIC-HH filter until therewas between 3-5 ml left in the syringe. The final amount of concentratedproduct was returned to the starting syringe by voiding the air from the10 ml syringe.

Example 8 Generation of Concentrated Products

For each patient, 4 preparations and 2 50 ml spin conical were madeaccording to the flow charts shown in the Figures. This allows for 6concentrated products for each patient. For preparation 1, an activatedproduct was made by mixing the RBCR portion with the PPP, thenconcentrating on either A) APIC-HL, or B) APIC-HH filter. In preparation2, the PPP was concentrated on either A) APIC-HL, or B) APIC-HH filterfirst, then mixed with the RBCR portion to generate an un-activatedproduct. Finally, preparation 3 the closest method to APIC preparationwas made by concentrating the plasma containing most of the plateletspulled from the 50 ml spin on either A) APIC-HL, or B) APIC-HH filter.

Cell Counting.

Red and white blood cells, and PPP samples were counted byhemacytometer. Platelets in all samples, except PPP, were counted bycoulter counter.

Volume.

The void volume for each filter in a large part determined how muchproduct was available for use after concentration in procedure 1. In theAPIC-HL, the void volume is 12 ml, however only an average of 3.5 ml isrecovered when the air is voided from the filter. The large void volumeprevents further concentration of the plasma and lowers the amount ofavailable product. The APIC-HH filter has a void volume of only 2 ml,where ˜1 ml is recovered in addition to what is concentrated in thesyringe. The average recovered from process 1B is 5 ml. Much moreproduct is available during process 2 because the RBCR is mixed afterthe fact, on average 9.6 ml and 10.5 ml for A and B respectively.Process 3 produces the approximately same volumes as process A givenonly the centrifugation is different (3.8 ml and 4 ml for A and Brespectively). A summary of the average volumes obtained at each stepare summarized in Table 2.

Cell Counts.

RBC and WBC were negligible for all products that went through the RBCRprocess. Those that did not (3A and 3B), had small amounts of RBC butelevated WBC (1-1.6 fold over whole blood). The highest concentration ofplatelets was seen in process 1 using the APIC filter (1A) for a 4.69×concentration over whole blood. Processes 1A, 2A and 2B had similarplatelet concentrations (3.14-3.97×). This was unexpected in processes1A and 2A. For process 1A, given the total volume concentrated only ˜2×,the platelets concentrated ˜3×. This may be an artifact of the lowpatient number. The platelet fold concentration and A2M foldconcentration is expected follow the volume concentrated to. In process2A where the high void volume in the APIC-HL (12 ml) would have dilutedthe RBCR (6-7 ml) and should have resulted in much lower plateletconcentrations. However, only 3-4 ml of product could be recovered fromthe APIC-HL, leading to a much smaller dilution factor. It should alsobe noted that in all processes, the RBCR intermediate is higher inplatelets than their corresponding concentrated products. This is due tothe removal of the RBC and WBC volumes, leaving more platelets in asmaller volume. The cell counts for platelets, RBC and WBC for eachproduct and intermediate are summarized in Table 3.

A2M Concentration.

A2M concentration was measured by ELISA for each PPP, whole blood, andconcentrated product. In all cases, the APIC filter concentrated A2Mbetter than the APIC-HL in each respective procedure. Pre-mixing withthe APIC-HH filter (1B) gave the most concentrated A2M products (3.44×compared to PPP), followed by premixing with the APIC-HL (1A) (2.67×compared to PPP). Both of these processes result in activated platelets.Processes 3B and 2B, both using the APIC filter, gave the next highestA2M concentrations (2.5× and 2.02×). Process 2A was the lowest A2Mconcentration, very close to plasma levels at 0.93×. This is the resultof the lower A2M concentration and lower volume recovery from theAPIC-HL where 3-4 ml of concentrated PPP is being mixed with the RBCR at6-7 ml. Therefore, use of an un-activated product would require theAPIC-HH filter to concentrate A2M above plasma levels.

TABLE 2 Avg Volume (mL) of Each Component for Each Process 1A 1B 2A 2B3A 3B Avg ± SD Avg ± SD Avg ± SD Avg ± SD Avg ± SD Avg ± SD ml PPP 21.0± 4.6 19.3 ± 4.5  19.7 ± 4.5 19.7 ± 3.8 n/a n/a ml PRP 10.2 ± 0.6 10.7 ±0.3  1.05 ± 0.0 10.5 ± 0.0 15.0 ± 0.0 15.0 ± 0.0  ml RBCR  6.6 ± 0.6 6.5± 0.7  5.8 ± 0.5  6.5 ± 1.6 n/a n/a total vol concentrated 27.6 ± 4.825.8 ± 4.2  19.5 ± 6.4 19.7 ± 3.8 n/a n/a ml in syringe  0.0 ± 0.0 4.0 ±1.0  0.0 ± 0.0  3.0 ± 0.0  0.0 ± 0.0 3.0 ± 0.0 ml added to RBCR n/a n/a 3.3 ± 0.4  3.3 ± 0.3 n/a n/a V_(f) produced concentrate 12.0 ± 0.0 6.0± 1.0 12.0 ± 0.0  5.0 ± 0.0 12.0 ± 0.0 5.0 ± 0.0 V_(f) recoveredconcentrate  3.5 ± 0.9 5.0 ± 1.0  3.5 ± 0.0  4.0 ± 0.0  3.8 ± 1.3 4.0 ±0.0 V_(f) total product recovered  3.5 ± 0.9 5.0 ± 1.0  9.6 ± 0.1 10.5 ±1.6  3.8 ± 1.3 4.0 ± 0.0 V_(f) total product produced 12.0 ± 0.0 6.0 ±1.0 15.1 ± 0.1 11.5 ± 1.6 12.0 ± 0.0 12.0 ± 0.0  produced = total amountestimated including void volumes, recovered = actual amount availableTable 2 legend - [ml PPP = amount of PPP taken off from Harvestcentrifugation, ml PRP = ml of platelet rich fraction harvested aftercentrifugation, ml RBCR = ml taken off of RBCR centrifugation, total volconcentrated = the total volume added to the filter for concentration,ml in syringe = amount of concentrate remaining in syringe when theconcentrate process is stopped, ml added to RBCR = ml of concentrateadded to the RBCR fraction, V_(f) produced concentrate = volume ofconcentrate produced, including what is not recoverable from the filter,V_(f) recovered concentrate = volume of concentrate that is recovered,and available after the air has been voided from the filter, V_(f) totalproduct recovered = the total amount of product recovered and available,including any RBCR added during the process, V_(f) total productproduced = amount of product produced including any amount not recoveredfrom the filter.]Table 2 legend—[ml PPP=amount of PPP taken off from Harvestcentrifugation, ml PRP=ml of platelet rich fraction harvested aftercentrifugation, ml RBCR=ml taken off of RBCR centrifugation, total volconcentrated=the total volume added to the filter for concentration, mlin syringe=amount of concentrate remaining in syringe when theconcentration process is stopped, ml added to RBCR=ml of concentrateadded to the RBCR fraction, V_(f) produced concentrate=volume ofconcentrate produced, including what is not recoverable from the filter,V_(f) recovered concentrate=volume of concentrate that is recovered andavailable after the air has been voided from the filter, V_(f) totalproduct recovered=the total amount of product recovered and available,including any RBCR added during the process, V_(f) total productproduced=amount of product produced including any amount not recoveredfrom the filter.]

TABLE 3 Cell Counts in Fold Change Over Whole Blood 1A 1B 2A 2B 3A 3Bsample AVE SD AVE SD AVE SD AVE SD AVE SD AVE SD Platelet WB 1.00 0.0001.00 0.000 1.00 0.000 1.00 0.000 1.00 0.000 1.00 0.000 PPP 0.48 0.0500.58 0.017 0.48 0.010 0.47 0.014 PRP 4.75 0.066 4.69 0.040 4.38 0.0184.57 0.023 1.70 0.024 1.70 0.024 RBCR 5.12 0.099 5.27 0.026 4.65 0.0584.75 0.024 CP 3.14 0.020 4.69 0.012 3.23 0.138 3.97 0.009 3.25 0.0394.54 0.025 RBC WB 1.00 0.000 1.00 0.000 1.00 0.000 1.00 0.000 1.00 0.0001.00 0.000 PPP 0.00 0.000 0.00 0.000 0.00 0.000 0.00 0.000 PRP 1.130.705 1.00 0.661 0.50 0.343 0.55 0.278 0.01 0.002 0.01 0.002 RBCR 0.000.000 0.00 0.001 0.00 0.000 0.00 0.000 CP 0.00 0.000 0.00 0.000 0.000.000 0.00 0.000 0.02 0.006 0.02 0.010 WBC WB 1.00 0.000 1.00 0.000 1.000.000 1.00 0.000 1.00 0.000 1.00 0.000 PPP 0.00 0.000 0.00 0.006 0.000.000 0.00 0.000 PRP 2.59 0.415 2.70 0.239 2.46 0.395 2.75 0.092 0.510.037 0.51 0.037 RBCR 0.01 0.005 0.14 0.224 0.02 0.017 0.02 0.023 CP0.01 0.007 0.00 0.000 0.02 0.030 0.00 0.000 1.06 0.184 1.65 0.019 *wholeblood (WB), platelet poor plasma (PPP), platelet rich plasma (PRP), redblood cell reduced (RBCR), concentrated product (CP)

Platelet Recovery.

Process 2B resulted in recoverable platelets at 60%, and process 2Aresulted in 51% platelet recovery. Both of these processes result inun-activated platelets and are due to the lower recovered concentratevolumes which are added to the RBCR intermediate to make the finalproduct. The APIC-HH filter had a greater percent platelet recovery foractivated products (processes 1 and 2) at 39% for process 1B and 30% forprocess 3B. In process 1B, there is ˜20% platelet loss at eachintermediate step. Moving closer to the buffy coat in the RBCRintermediate would increase the recovery in that step some. For process3B, the largest loss of platelets is due to the inefficiency of thecentrifugation step. The APIC-HL had lower platelet recovery of theactivated processes (1A and 3A), potentially due to the large amount ofvolume remaining on the filter.

Example 9 System Overview

The APIC PRP System (FIGS. 24-30) can contain three components forproducing APIC PRP; High Speed Bench Top Centrifuge; Peristaltic Pump w/Custom Housing; and Disposables Kit for Collection, Separation, andAdministration of APIC PRP. The APIC System can separate and concentratea patient's own blood for therapeutic use by a physician. 60 cc to 120cc of a patient's blood can be drawn in to a collection bag, thentransferred to centrifuge tubes. The tubes can be centrifuged and therecovered plasma is then drawn off and transferred to a concentrationbag. The pump can circulate the blood through a Tangential Flow Filterconcentrating the APIC PRP down to a 5 cc to 10 cc of APIC. The APIC canthen be used by Physicians as they deem necessary and appropriate. Thesystem can include: Industry Standard Centrifuge and Peristaltic Pump,Private Labeled and Customized for APIC, Low Cost Disposable withFiltration, Majority of Disposable Components are PPS, Minimal number ofsteps. The system can include: Integrated Centrifuge and PumpSeparation, Custom Ergonomic Design, Lower Cost Equipment w/SmallerFootprint, Lower Cost and Less Disposables, Ease Of Use=Set It AndForget It

APIC Cell Free Concentration Kit: No Centrifugation; Direct Connectionof Blood Collection Bag to Concentration Bag; Two Filters

Example 10 Preparation of Blood for Autologous Therapy

120 mL of whole human blood was obtained from a subject by venipuncture.38 mL aliquots of the blood were collected into two or more hematologiccollection bottles with a suitable volume of citrate dextrose solution A(“ACD-A”) in each collection bottle. The collection bottles withblood/ACD-A were placed into a fixed angle rotor centrifuge, andcentrifuged at predetermined velocities and times under ambienttemperature conditions. Approximately 15 mL of plasma was aliquoted fromeach tube with a serological pipette, leaving approximately 1 mL, ofplasma above the level of the buffy coat so as not to disturb theprecipitated cells. This process was repeated for the collection bottlesin one or more centrifuge spin cycles to yield a volume 45 mL of totalplasma from a total blood draw of 120 mL. The plasma was pooled into aseparate sterile hematologic collection bag. The compositions describedherein can be mixed with pharmaceutical acceptable excipients, beforeadministration to a subject.

Example 11 Inhibition of ADAMTS-5- and ADAMTS-4- with A2M

Bovine Cartilage Explants (BCEs) were treated with 500 ng/ml ADAMTS-5 orADAMTS-4 for 2 days, with a 3-fold serial dilution of purified A2M.Concentration of A2M tested were 100, 33.3, 11.1, 3.7, 1.2, 0.4 μg/mL.The A2M inhibited the proteases in a concentration dependent manner. TheIC₅₀ for inhibiting 500 ng/ml of ADAMTS-5 was calculated to be ˜7 μg/mlA2M (a 1:1 molar ratio). Maximum inhibition was observed in ˜90% with100 μg/ml A2M (a 14:1 molar ratio). The A2M was shown to block formationof Aggrecan G3 fragments and FAC formation.

Example 12 Comparison of APIC Retentate and Filtrate

Wounds are treated with the A2M compositions containing ˜7 mg/ml A2M.Wound healing is efficiently promoted by 1% v/v of the Retentate of theA2M compositions (concentration of proteins >500 kDa in size), but notby the filtrate (contains proteins <500 kDa), even at 5% v/v. Theenhanced wound healing effects are dose dependent. The inability offiltrate to protect cartilage from catabolism by ADAMTS-5 demonstratesthat the APIC system concentrates >99% of the protective factors ofautologous blood.

Example 13 Cytokine Profile of Wound Cells Treated with APIC

Wound cells are treated with or without APIC or recombinant A2M for 2days and the activation of the wound cells is monitored by secretion ofcytokines and growth factors into the wound. Wound cells do not show achange in the cytokine profile of the test.

Example 14 Wound Healing Effect in Rabbit Model

The ability of the Autologous Protease Inhibitor Concentrate (APIC-CellFree), which contains concentrated A2M from the blood, or recombinantA2M, is tested to promote wound healing, in a rabbit model. The rabbitmodel represents a functional load-bearing in vivo anatomical model forthe evaluation of wound healing, which exhibits mechanical properties,morphological structures, and healing capacity similar to human tissues.Female 8-12 months old New Zealand white rabbits are used. Group 1: 6rabbits receive a wound on the right knee and a wound on the left knee.Applications of recombinant A2M or Autologous Protease InhibitorConcentrate (APIC-Cell Free) are prepared from the rabbit blood, andadministered to the wound on the right knee one or more days followingthe wound and saline solution (sham) is administered to the wound on theleft knee one or more days following the wound.

Autologous A2M Concentrate Preparation

Prior to the wound, 20 mL of blood is removed from each animal in group1 and is used to prepare the APIC Cell Free concentrate using a seriesof filters. Every rabbit receives the protease inhibitor concentratefrom its own blood. After treating the wound, the animal is sacrificedfor macroscopic and microscopic wound healing evaluation to determinehealing advancement and enhancement.

Macroscopic and Histological Analyses

For macroscopic evaluation, the wound surfaces are analyzed. Aftermacroscopic examination, wound samples were analyzed for histological(microscopic) evaluation. Macroscopic evaluation of the wounddemonstrates features consistent with enhanced wound healing. Treatmentwith APIC Cell Free or recombinant A2M considerably improves woundappearance. Application of APIC enhances wound healing by 53+/−20%compared to untreated controls (mean±SEM. p=0.0086). The concentrationof A2M in the APIC Cell Free varies front 5-65 mg/ml. There is adose-dependent correlation between higher concentrations of A2M in theAPI Cell Free and enhancement of wound healing on the macroscopicevaluation. The data suggests that the Autologous Protease InhibitorConcentrate (APIC-Cell Free), which contains 9-10 times the A2Mconcentration in blood, has an enhanced wound healing effect on a rabbitmodel.

Example 15 In Vitro Effect of A2M on Wound Healing

To test the hypothesis that the addition of proinflammatory cytokines orcartilage-degrading metalloproteinases (ADAMTS and MMP) slow woundhealing that will be inhibited by recombinant or autologous A2M, acontrolled in vitro wound healing assay is performed. Cells from animalwounds are treated with or without proinflammatory cytokines (TNF-α orIL-1β) or cartilage-degrading metalloproteinases (ADAMTS-5, ADAMTS-4,MMP-7, or MMP-12) in the presence or absence of recombinant orautologous A2M compositions. Wound cells are incubated 2 days inSerum-Free Media (SFM) with or without 500 ng/mL ADAMTS-4 or ADAMTS-5and 3-5 μg/mL of MMP-3, MMP-7, MMP-12, or MMP-13. MMP-3 is activatedwith chymotrypsin before application on wound cells. Forcytokine-induced retardation of wound healing, wound cells are incubated3 days in SFM with or without 80 ng/ml human TNF-α and 8 ng/mL human1L-1β. Wound healing is enhanced with the addition of 100 μg/mL ofpurified human recombinant or autologous A2M for protease digestion or 5mg/mL recombinant or autologous A2M for cytokine-induced degradation.

Example 16 Determination of Collagenase Activity and Inhibition by A2MCompositions

Reagents/Materials

140731NL Wound Fluid (15.34 μg/ml collagenase)

FITC-collagen, Type I (Life Technologies, D12060)

Purified plasma A2M (Molecular Innovations, HA2MG)

1×TN buffer (50 mM Tris pH 7.5, 150 mM NaCl)

10×TNCB (500 mM Tris pH7.5, 1.5M NaCl, 100 mM CaCl2, 0.2% NaN3, 0.5%Brijj35)

A2M (Molecular Innovations)

Sample Preparation

The wound fluid sample was diluted to prepare the 10×Cf Dil (1:40) bymixing 6.5 ul with 254 ul TN on ice (Vf 260 ul). A 1/400 dilution wasthe working concentration. Serially diluted A2M samples were prepared bymixing 76.8 ul of the purified plasma A2M (4.163 mg/ml) with 3.1 ul ofTN (Vf 80 ul, Cf 4000 μg/ml) to create sample 1. As shown in the tablebelow, 30 ul of this sample (1) was then added to 60 ul of TN (Vf 90 ul,Cf 1333 μg/ml) to create sample 2. 30 ul of this sample (2) was thenadded to 60 ul of TN (Vf 90 ul, Cf 444 μg/ml) to create sample 3. 30 ulof this sample (3) was then added to 60 ul of TN (Vf 90 ul, Cf 148μg/ml) to create sample 4. 30 of this sample (4) was then added to 60 ulof TN (Vf 90 ul, Cf 49.4 μg/ml) to create sample 5. 30 ul of this sample(5) was then added to 60 ul of TN (Vf 16.5 ul, Cf 1333 μg/ml) to createsample 6.

1 2 3 4 5 6 Transfer (μL) 30 30 30 30 30 30 1x TN (μl) 80 60 60 60 60 6010x Cf dil 4000 1333 444 148 49.4 16.5 Work Cf Dil 400 133 44.4 14.8 4917Digestion

160 ul FITC-collagen was diluted 1:1 with 160 ul TN (Vf 350 μl) and keptin the dark. The following were combined in a 96-well black plate.

Col - Only WF WF + A2M Vf (μl) WF (μl) 0 20 20 200 10x A2M (μl) 0 0 20200 10x TNCB (μl) 20 20 20 200 WFI H₂O (μl) 160 140 120 200 FITC-Col(μl) 20 20 20 200

The microplate was then read using a spectrophotometer at the excitationwavelength of 495 nm and emission wavelength of 515 nm every 20 secondinterval for 30 mins.

Example 17 Wound Fluid Collection Technique

There are several techniques that were utilized to collect wound fluid.One technique involved aspirating wound fluid from wet wounds utilizinga syringe. Another technique involved use of a filter paper to absorbthe wound fluid, followed by extraction of the absorbed wound fluid fromthe filter paper, such as by washing with a buffer. Another techniqueinvolved running a straight edge tongue blade across the wound andcollecting the fluid that gathered in front of the straight edge, suchas with a filter paper.

For example, human chronic wound fluid is extracted from primary woundfluid dressing by soaking a single dressing overnight in 5 ml phosphatebuffered saline pH 4.0-6.0 50 mM sodium acetate adjusted to relevant pHwith glacial acetic buffer acid pH 7.0-8.0 0.2MTris(hydroxymethyl)aminomethane (Tris) corrected to buffer relevant pHusing 0.2M hydrochloric acid.

Example 18 Effects of A2M Compositions on Wound Healing in Diabetic Rats

Summary

Healing of chronic wounds such as diabetic ulcers is a significantclinical problem. This study examines the in vivo response to thetherapeutic recombinant or autologous compositions according to thepresent invention. The preliminary animal study on a diabetic rat modelwith impaired wound healing is conducted comparing the recombinant orautologous A2M compositions described herein with distilled water. As aresult, the time to complete closure of wounds is lower in the A2Mtreated group. The difference in wound healing since day 9^(th) of thetreatment is apparent. The A2M treated animals have lower scar tissuesand the fur growth is complete. In water-treated animals a scar withimpaired fur growth is apparent. The results of this study suggest thatdermal use of these A2M compositions have a potential to modulate woundhealing and stimulate fur growth.

Methods

The animal model for in vivo testing of the recombinant or autologousA2M compositions is a full-thickness wound in the dorsal skin ofdiabetic rats. Wistar rats weighing 200-250 g are used. Animals arecaged in separate cages, Diabetes is induced by administration ofstreptozotocin (Sigma-Aldrich, UK). Streptozotocin is administered atdose of 55 mg/kg intraperitoneally. Before the administration ofstreptozotocin, a baseline blood glucose of rats is determined. After 48hours, the blood glucose is again measured to ensure rats are diabetic.The induction of diabetes is confirmed if the blood glucose level isdoubled. Glucose is determined by a Glucometer (Infopia Co., Korea).Determination of blood glucose continues every 5 days to ensure thesubsistence of diabetes. Regarding the entity of streptozotocin-induceddiabetes, the animals which lose much weight and become week, and thosewith uncertain blood glucose levels are excluded from the study. A totalof 14 rats are used with equal numbers in control and test groups. Thetest group has a volume of a solution comprising the recombinant orautologous A2M composition applied and the control group is dressed withdistilled water. At time=0 days, a full-thickness, circular 15 mmdiameter wound is created (e.g., according to Wound Rep. Reg. 2002; 10:286-294). Rats are anaesthetized by intraperitoneal pentobarbital (55mg/kg) and the dorsal skin is prepared for surgery using Betadine. Thewound is created using surgical scissors. At time=0 days dressings areplaced, as prepared, directly on the wounds. The wounds are covered bysterile gases and wrapped carefully. Every 2-3 days following surgery,wounds were redressed with fresh control or test dressings while therats were under anesthesia. The wounds are flushed with sterile salineto remove debris and to clean the wound area. A digital camera is usedto take the pictures of the wound. The pictures are examined for woundhealing in terms of wound size and appearance of new fresh epithelium.Once photographed, fresh dressings are placed on the wounds, and thewounds are covered again. Control of bias is achieved by assigning acode to each of the experimental groups. Investigators are blinded tothe identity of each of the groups and the test and control have asimilar appearance. The code is broken following completion of the final4-week analysis.

In the test group on the 15^(th) day of therapy the wound is completelyclosed and the new, short fur covers the scar area. On the 22^(nd) dayof therapy the wound is completely healed and the new, long fur coversthe entire scar area. No signs of the previous wound can be seen. In thecontrol group on the 15^(th) day of therapy the wound is not closed. Onthe 22^(nd) day of testing the wound is closed but the scar is stillsever and completely naked.

Wound areas and perimeters are similar in test and control groups;however, there is a tendency for more rapid closure in the test group,particularly at day 15 where the difference in wound areas andperimeters is most pronounced. The time to complete closure of wounds islower in A2M treated animals. In both control and test groups, woundarea begins to decrease at day 9^(th) and approximately complete woundclosure first occurs by day 15^(th) (one out of seven rats). By day22^(nd), are essentially closed in both groups but growth of fur in theA2M treated group is especially complete as compared to thewater-treated group.

The results of this study suggest that dermal preparation comprising therecombinant or autologous A2M compositions according to the presentinvention has potential to enhance wound healing. In addition toaccelerating wound closure, A2M treatment in this study appears toimprove the quality of the tissue in the healing wound since the furgrew more efficiently than in the control group. Chronic wounds are notonly characterized by untimely healing and the inability to remainclosed following healing. Thus, time to closure may not be the onlyrelevant end point or sole basis for efficacy of the treatment.Obtaining the healthier scar tissue in the test group animals treatedwith the recombinant or autologous A2M compositions allows anticipatinga lowered recurrence rate.

Example 19 Wound Debridement

Recombinant or autologous A2M compositions are applied to necrotictissues on pigs for an in vivo debridement efficacy study. Recombinantor autologous A2111 compositions, together with a debrider, are used toeach of the wounds generated (about 2 cm in diameter). After 24 hours,significant wound debridement is observed on the wounds treated with theA2M compositions. After 5 days, those with recombinant or autologous A2Mcompositions show clean surfaces without any necrotic tissue andcomplete healing. Debrider treated wounds also show significantdebridement after 48 hours. However, the wounds are not as clean asthose treated with recombinant or autologous A2M compositions, and didnot show complete healing after five days.

Example 20 Sequences of Modified Recombinant A2M Bait Regions

Sequences:

SEQ ID NO 1: Wild-Type A2M Precursor Protein—Complete Vector DNASequence Including Tag Sequences for Easier Purification.

1 CTCATGACCA AAATCCCTTA ACGTGAGTTA CGCGCGCGTC GTTCCACTGA GCGTCAGACC

61 CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT

121 TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA

181 CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG

241 TGTAGCCGTA GTTAGCCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC

301 TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG

361 ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA

421 CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT

481 GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG

541 TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC

601 CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC

661 GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC

721 CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG

781 CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA

841 GCGAGGAAGC GGAAGGCGAG AGTAGGGAAC TGCCAGGCAT CAAACTAAGC AGAAGGCCCC

901 TGACGGATGG CCTTTTTGCG TTTCTACAAA CTCTTTCTGT GTTGTAAAAC GACGGCCAGT

961 CTTAAGCTCG GGCCCCCTGG GCGGTTCTGA TAACGAGTAA TCGTTAATCC GCAAATAACG

1021 TAAAAACCCG CTTCGGCGGG TTTTTTTATG GGGGGAGTTT AGGGAAAGAG CATTTGTCAG

1081 AATATTTAAG GGCGCCTGTC ACTTTGCTTG ATATATGAGA ATTATTTAAC CTTATAAATG

1141 AGAAAAAAGC AACGCACTTT AAATAAGATA CGTTGCTTTT TCGATTGATG AACACCTATA

1201 ATTAAACTAT TCATCTATTA TTTATGATTT TTTGTATATA CAATATTTCT AGTTTGTTAA

1261 AGAGAATTAA GAAAATAAAT CTCGAAAATA ATAAAGGGAA AATCAGTTTT TGATATCAAA

1321 ATTATACATG TCAACGATAA TACAAAATAT AATACAAACT ATAAGATGTT ATCAGTATTT

1381 ATTATCATTT AGAATAAATT TTGTGTCGCC CTTAATTGTG AGCGGATAAC AATTACGAGC

1441 TTCATGCACA GTGGCGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG

1501 GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC

1561 CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA

1621 TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG

1681 CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG

1741 ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT

1801 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA

1861 TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG

1921 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT

1981 CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG

2041 CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT ACGACTCACT

2101 ATAGGGGTAC CTGCCACCAT GGGGAAAAAC AAACTGCTGC ATCCAAGCCT GGTCCTGCTG

2161 CTGCTGGTTC TGCTGCCTAC TGACGCCTCT GTGAGCGGAA AGCCCCAGTA TATGGTTCTG

2221 GTCCCGTCCC TGCTGCACAC CGAGACCACA GAAAAAGGGT GCGTGCTGCT GTCTTACCTG

2281 AATGAAACAG TGACTGTTAG TGCCTCACTG GAGAGTGTGC GCGGAAATCG TTCACTGTTC

2341 ACCGATCTGG AGGCGGAAAA CGATGTGCTG CATTGCGTCG CATTTGCTGT GCCAAAAAGC

2401 TCCTCTAATG AAGAAGTGAT GTTCCTGACC GTCCAGGTGA AGGGCCCTAC ACAGGAATTC

2461 AAAAAACGCA CTACCGTTAT GGTCAAAAAC GAGGATAGCC TGGTGTTTGT TCAGACAGAC

2521 AAATCCATCT ATAAGCCTGG TCAGACTGTG AAGTTCCGGG TGGTTAGCAT GGATGAAAAT

2581 TTTCACCCCC TGAACGAGCT GATTCCACTG GTGTACATCC AGGACCCTAA AGGCAACCGC

2641 ATCGCCCAGT GGCAGTCTTT CCAGCTGGAA GGCGGTCTGA AGCAGTTTAG TTTCCCTCTG

2701 AGTTCAGAGC CGTTTCAGGG TTCTTATAAA GTCGTGGTTC AGAAAAAGAG TGGGGGACGT

2761 ACTGAACATC CTTTTACCGT TGAAGAGTTC GTCCTGCCGA AATTTGAGGT CCAGGTGACC

2821 GTTCCCAAGA TTATCACAAT TCTGGAAGAG GAAATGAACG TGAGCGTGTG CGGACTGTAT

2881 ACCTACGGCA AACCAGTGCC TGGTCACGTT ACAGTCAGTA TCTGCCGTAA GTACTCAGAT

2941 GCAAGCGACT GTCATGGCGA AGATTCACAG GCTTTTTGCG AGAAGTTCAG CGGCCAGCTG

3001 AACTCCCACG GTTGCTTCTA TCAGCAGGTG AAAACCAAGG TTTTTCAGCT GAAACGGAAG

3061 GAGTACGAAA TGAAACTGCA TACAGAAGCC CAGATTCAGG AAGAAGGCAC CGTCGTGGAA

3121 CTGACTGGTC GTCAGAGCTC CGAGATTACC CGGACAATCA CTAAACTGAG CTTCGTGAAG

3181 GTTGATTCCC ACTTTCGGCA GGGGATTCCC TTTTTCGGAC AGGTGCGCCT GGTTGACGGG

3241 AAAGGAGTTC CGATCCCCAA CAAAGTGATC TTTATTCGCG GCAATGAAGC CAACTATTAC

3301 AGCAACGCGA CAACTGATGA GCATGGGCTG GTGCAGTTCA GTATCAATAC CACAAACGTG

3361 ATGGGAACCT CACTGACAGT CCGCGTGAAT TATAAAGACC GTTCACCGTG TTATGGCTAC

3421 CAGTGGGTGA GCGAGGAACA CGAGGAAGCC CACCATACCG CGTACCTGGT TTTCAGCCCC

3481 TCCAAATCTT TTGTCCATCT GGAACCTATG TCTCACGAGC TGCCGTGCGG CCATACCCAG

3541 ACAGTGCAGG CACATTATAT TCTGAACGGC GGCACCCTGC TGGGTCTGAA AAAGCTGAGC

3601 TTTTATTACC TGATTATGGC TAAGGGGGGA ATCGTCCGCA CTGGCACCCA CGGTCTGCTG

3661 GTTAAACAGG AAGATATGAA GGGCCATTTC AGTATTTCAA TCCCTGTTAA AAGCGACATT

3721 GCTCCGGTCG CCCGTCTGCT GATCTATGCC GTGCTGCCAA CCGGCGATGT TATCGGTGAC

3781 TCCGCCAAAT ACGATGTGGA GAATTGTCTG GCGAACAAGG TTGACCTGAG CTTTTCCCCC

3841 TCTCAGAGTC TGCCAGCGTC TCATGCACAT CTGCGTGTGA CCGCAGCCCC TCAGAGCGTT

3901 TGCGCTCTGC GTGCAGTGGA TCAGTCCGTG CTGCTGATGA AGCCAGACGC AGAACTGTCT

3961 GCTAGCAGCG TGTATAATCT GCTGCCTGAG AAAGATCTGA CCGGGTTCCC AGGACCTCTG

4021 AACGATCAGG ATGACGAAGA CTGTATTAAT CGCCACAACG TGTATATTAA TGGGATCACA

4081 TACACTCCGG TTTCAAGCAC CAACGAAAAA GATATGTACA GCTTCCTGGA GGACATGGGT

4141 CTGAAAGCGT TTACCAATTC CAAGATCCGG AAACCCAAGA TGTGCCCACA GCTGCAGCAG

4201 TATGAAATGC ACGGACCTGA GGGTCTGCGT GTGGGCTTTT ACGAATCTGA TGTGATGGGA

4261 CGTGGTCATG CACGTCTGGT TCATGTCGAG GAACCACACA CCGAAACAGT GCGTAAATAC

4321 TTCCCTGAGA CCTGGATTTG GGACCTGGTT GTGGTGAACT CCGCGGGTGT GGCAGAAGTG

4381 GGTGTTACCG TCCCGGATAC TATTACCGAA TGGAAAGCAG GTGCCTTCTG TCTGTCTGAG

4441 GATGCAGGGC TGGGAATCTC CTCTACAGCC TCTCTGCGCG CGTTTCAGCC CTTTTTCGTC

4501 GAACTGACTA TGCCATATAG CGTGATTCGT GGCGAGGCAT TCACTCTGAA AGCTACCGTG

4561 CTGAATTACC TGCCCAAGTG CATCCGCGTG AGCGTGCAGC TGGAAGCTAG TCCCGCCTTT

4621 CTGGCGGTCC CAGTGGAGAA GGAACAGGCA CCGCACTGCA TTTGTGCTAA CGGCCGGCAG

4681 ACTGTTTCCT GGGCCGTCAC CCCCAAATCT CTGGGTAATG TGAACTTCAC CGTTTCAGCA

4741 GAGGCTCTGG AAAGCCAGGA GCTGTGCGGC ACCGAAGTCC CATCCGTGCC TGAGCATGGT

4801 CGCAAAGATA CAGTCATCAA GCCTCTGCTG GTTGAACCGG AAGGCCTGGA GAAGGAAACT

4861 ACCTTTAATT CTCTGCTGTG CCCAAGTGGC GGTGAAGTGT CCGAGGAACT GTCTCTGAAA

4921 CTGCCGCCCA ACGTGGTCGA GGAATCTGCC CGTGCGTCAG TTAGCGTCCT GGGGGATATT

4981 CTGGGAAGTG CCATGCAGAA TACCCAGAAC CTGCTGCAGA TGCCGTATGG CTGTGGCGAG

5041 CAGAATATGG TTCTGTTTGC GCCCAACATC TATGTCCTGG ATTACCTGAA TGAAACACAG

5101 CAGCTGACTC CTGAAATCAA AAGCAAGGCA ATCGGGTATC TGAATACCGG ATACCAGCGG

5161 CAGCTGAACT ATAAGCACTA CGACGGCTCC TATTCTACCT TCGGCGAACG GTACGGTCGC

5221 AATCAGGGGA ACACTTGGCT GACCGCCTTT GTGCTGAAAA CCTTTGCCCA GGCTCGCGCC

5281 TATATCTTTA TTGATGAGGC CCATATTACA CAGGCGCTGA TCTGGCTGTC ACAGCGCCAG

5341 AAGGACAACG GGTGTTTCCG TAGTTCAGGA AGCCTGCTGA ACAATGCCAT CAAAGGCGGC

5401 GTCGAGGATG AAGTGACACT GAGCGCATAC ATTACTATCG CTCTGCTGGA AATCCCTCTG

5461 ACAGTGACTC ACCCGGTGGT TCGCAATGCT CTGTTTTGCC TGGAAAGTGC ATGGAAAACA

5521 GCTCAGGAAG GCGATCACGG ATCACACGTG TATACTAAGG CACTGCTGGC GTACGCATTC

5581 GCTCTGGCCG GCAACCAGGA TAAACGTAAA GAAGTGCTGA AATCACTGAA TGAGGAAGCA

5641 GTTAAAAAGG ACAACAGCGT CCACTGGGAA CGGCCGCAGA AACCCAAGGC TCCAGTGGGT

5701 CACTTTTATG AGCCTCAGGC ACCGAGTGCT GAGGTGGAAA TGACCTCATA TGTTCTGCTG

5761 GCATACCTGA CCGCACAGCC TGCCCCCACA TCAGAAGATC TGACAAGCGC CACTAATATT

5821 GTGAAATGGA TCACCAAGCA GCAGAACGCG CAGGGCGGTT TTAGCTCCAC CCAGGACACA

5881 GTCGTGGCAC TGCACGCTCT GTCTAAATAT GGGGCAGCTA CCTTCACACG CACTGGAAAG

5941 GCCGCGCAAG TGACTATTCA GTCTAGTGGC ACCTTTTCAA GCAAGTTCCA GGTGGATAAC

6001 AATAACCGTC TGCTGCTGCA GCAGGTGTCC CTGCCCGAAC TGCCAGGCGA GTACTCTATG

6061 AAAGTCACTG GGGAAGGATG CGTGTATCTG CAGACCTCCC TGAAATACAA TATTCTGCCC

6121 GAGAAAGAAG AATTTCCATT CGCACTGGGC GTGCAGACCC TGCCTCAGAC ATGCGATGAA

6181 CCGAAGGCTC ATACTTCTTT TCAGATCAGT CTGTCAGTGA GCTATACCGG GTCCCGCTCT

6241 GCCAGTAACA TGGCGATTGT GGATGTGAAA ATGGTGAGTG GATTCATCCC TCTGAAACCG

6301 ACTGTGAAGA TGCTGGAACG GAGTAATCAC GTTTCACGCA CCGAGGTCTC CTCTAACCAT

6361 GTGCTGATCT ACCTGGATAA AGTGTCCAAT CAGACACTGT CTCTGTTTTT CACTGTGCTG

6421 CAGGATGTCC CCGTGCGTGA CCTGAAACCA GCCATTGTTA AGGTCTATGA TTATTACGAA

6481 ACCGACGAGT TCGCGATCGC AGAATACAAC GCGCCGTGCA GCAAAGACCT GGGGAATGCT

6541 GACTACAAGG ACGACGACGA CAAGGGGGCA AGCCACCACC ATCACCATCA CTAAGGATCC

6601 AAAATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT

6661 GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT

6721 TGCATCACAA CACTCAACCC TATCTCGGTC TATTCTTTTG ATTTATAAGG GATTTTGCCG

6781 ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA AATTTAACGC GAATTAATTC

6841 TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA

6901 TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG

6961 CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA

7021 CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC

7081 TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCTGC CTCTGAGCTA TTCCAGAAGT

7141 AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA GCTCCCGGGA GCTTGTATAT

7201 CCATTTTCGG ATCTGATCAG CACGTGTTGA CAATTAATCA TCGGCATAGT ATATCGGCAT

7261 AGTATAATAC GACAAGGTGA GGAACTAAAC CATGGCCAAG CCTTTGTCTC AAGAAGAATC

7321 CACCCTCATT GAAAGAGCAA CGGCTACAAT CAACAGCATC CCCATCTCTG AAGACTACAG

7381 CGTCGCCAGC GCAGCTCTCT CTAGCGACGG CCGCATCTTC ACTGGTGTCA ATGTATATCA

7441 TTTTACTGGG GGACCTTGTG CAGAACTCGT GGTGCTGGGC ACTGCTGCTG CTGCGGCAGC

7501 TGGCAACCTG ACTTGTATCG TCGCGATCGG AAATGAGAAC AGGGGCATCT TGAGCCCCTG

7561 CGGACGGTGC CGACAGGTGC TTCTCGATCT GCATCCTGGG ATCAAAGCCA TAGTGAAGGA

7621 CAGTGATGGA CAGCCGACGG CAGTTGGGAT TCGTGAATTG CTGCCCTCTG GTTATGTGTG

7681 GGAGGGCTAA CACGTGCTAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG

7741 CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT

7801 GGAGTTCTTC GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA

7861 TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC

7921 CAAACTCATC AATGTATCTT ATCATGTCTG TATACCGTCG ACCTCTAGCT AGAGCTTGGC

7981 GTAATCATGG TCATTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT

8041 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT

8101 TACCATCTGG CCCCAGCGCT GCGATGATAC CGCGAGAACC ACGCTCACCG GCTCCGGATT

8161 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT

8221 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA

8281 ATAGTTTGCG CAACGTTGTT GCCATCGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG

8341 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT

8401 TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG

8461 CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG

8521 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC

8581 GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA

8641 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC

8701 CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT

8761 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG

8821 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATATTCTT CCTTTTTCAA TATTATTGAA

8881 GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA

8941 AACAAATAGG GGTCAGTGTT ACAACCAATT AACCAATTCT GAACATTATC GCG

SEQ ID NO 2: Complete Vector DNA Sequence of the of the Acceptor Mutant.

1 CTCATGACCA AAATCCCTTA ACGTGAGTTA CGCGCGCGTC GTTCCACTGA GCGTCAGACC

61 CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT

121 TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA

181 CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG

241 TGTAGCCGTA GTTAGCCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC

301 TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG

361 ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA

421 CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT

481 GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG

541 TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC

601 CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC

661 GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC

721 CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG

781 CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA

841 GCGAGGAAGC GGAAGGCGAG AGTAGGGAAC TGCCAGGCAT CAAACTAAGC AGAAGGCCCC

901 TGACGGATGG CCTTTTTGCG TTTCTACAAA CTCTTTCTGT GTTGTAAAAC GACGGCCAGT

961 CTTAAGCTCG GGCCCCCTGG GCGGTTCTGA TAACGAGTAA TCGTTAATCC GCAAATAACG

1021 TAAAAACCCG CTTCGGCGGG TTTTTTTATG GGGGGAGTTT AGGGAAAGAG CATTTGTCAG

1081 AATATTTAAG GGCGCCTGTC ACTTTGCTTG ATATATGAGA ATTATTTAAC CTTATAAATG

1141 AGAAAAAAGC AACGCACTTT AAATAAGATA CGTTGCTTTT TCGATTGATG AACACCTATA

1201 ATTAAACTAT TCATCTATTA TTTATGATTT TTTGTATATA CAATATTTCT AGTTTGTTAA

1261 AGAGAATTAA GAAAATAAAT CTCGAAAATA ATAAAGGGAA AATCAGTTTT TGATATCAAA

1321 ATTATACATG TCAACGATAA TACAAAATAT AATACAAACT ATAAGATGTT ATCAGTATTT

1381 ATTATCATTT AGAATAAATT TTGTGTCGCC CTTAATTGTG AGCGGATAAC AATTACGAGC

1441 TTCATGCACA GTGGCGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG

1501 GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC

1561 CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA

1621 TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG

1681 CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG

1741 ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT

1801 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA

1861 TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG

1921 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT

1981 CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG

2041 CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT ACGACTCACT

2101 ATAGGGGTAC CTGCCACCAT GGGGAAAAAC AAACTGCTGC ATCCAAGCCT GGTCCTGCTG

2161 CTGCTGGTTC TGCTGCCTAC TGACGCCTCT GTGAGCGGAA AGCCCCAGTA TATGGTTCTG

2221 GTCCCGTCCC TGCTGCACAC CGAGACCACA GAAAAAGGGT GCGTGCTGCT GTCTTACCTG

2281 AATGAAACAG TGACTGTTAG TGCCTCACTG GAGAGTGTGC GCGGAAATCG TTCACTGTTC

2341 ACCGATCTGG AGGCGGAAAA CGATGTGCTG CATTGCGTCG CATTTGCTGT GCCAAAAAGC

2401 TCCTCTAATG AAGAAGTGAT GTTCCTGACC GTCCAGGTGA AGGGCCCTAC ACAGGAATTC

2461 AAAAAACGCA CTACCGTTAT GGTCAAAAAC GAGGATAGCC TGGTGTTTGT TCAGACAGAC

2521 AAATCCATCT ATAAGCCTGG TCAGACTGTG AAGTTCCGGG TGGTTAGCAT GGATGAAAAT

2581 TTTCACCCCC TGAACGAGCT GATTCCACTG GTGTACATCC AGGACCCTAA AGGCAACCGC

2641 ATCGCCCAGT GGCAGTCTTT CCAGCTGGAA GGCGGTCTGA AGCAGTTTAG TTTCCCTCTG

2701 AGTTCAGAGC CGTTTCAGGG TTCTTATAAA GTCGTGGTTC AGAAAAAGAG TGGGGGACGT

2761 ACTGAACATC CTTTTACCGT TGAAGAGTTC GTCCTGCCGA AATTTGAGGT CCAGGTGACC

2821 GTTCCCAAGA TTATCACAAT TCTGGAAGAG GAAATGAACG TGAGCGTGTG CGGACTGTAT

2881 ACCTACGGCA AACCAGTGCC TGGTCACGTT ACAGTCAGTA TCTGCCGTAA GTACTCAGAT

2941 GCAAGCGACT GTCATGGCGA AGATTCACAG GCTTTTTGCG AGAAGTTCAG CGGCCAGCTG

3001 AACTCCCACG GTTGCTTCTA TCAGCAGGTG AAAACCAAGG TTTTTCAGCT GAAACGGAAG

3061 GAGTACGAAA TGAAACTGCA TACAGAAGCC CAGATTCAGG AAGAAGGCAC CGTCGTGGAA

3121 CTGACTGGTC GTCAGAGCTC CGAGATTACC CGGACAATCA CTAAACTGAG CTTCGTGAAG

3181 GTTGATTCCC ACTTTCGGCA GGGGATTCCC TTTTTCGGAC AGGTGCGCCT GGTTGACGGG

3241 AAAGGAGTTC CGATCCCCAA CAAAGTGATC TTTATTCGCG GCAATGAAGC CAACTATTAC

3301 AGCAACGCGA CAACTGATGA GCATGGGCTG GTGCAGTTCA GTATCAATAC CACAAACGTG

3361 ATGGGAACCT CACTGACAGT CCGCGTGAAT TATAAAGACC GTTCACCGTG TTATGGCTAC

3421 CAGTGGGTGA GCGAGGAACA CGAGGAAGCC CACCATACCG CGTACCTGGT TTTCAGCCCC

3481 TCCAAATCTT TTGTCCATCT GGAACCTATG TCTCACGAGC TGCCGTGCGG CCATACCCAG

3541 ACAGTGCAGG CACATTATAT TCTGAACGGC GGCACCCTGC TGGGTCTGAA AAAGCTGAGC

3601 TTTTATTACC TGATTATGGC TAAGGGGGGA ATCGTCCGCA CTGGCACCCA CGGTCTGCTG

3661 GTTAAACAGG AAGATATGAA GGGCCATTTC AGTATTTCAA TCCCTGTTAA AAGCGACATT

3721 GCTCCGGTCG CCCGTCTGCT GATCTATGCC GTGCTGCCAA CCGGCGATGT TATCGGTGAC

3781 TCCGCCAAAT ACGATGTGGA GAATTGTCTG GCGAACAAGG TTGACCTGAG CTTTTCCCCC

3841 TCTCAGAGTC TGCCAGCGTC TCATGCACAT CTGCGTGTGA CCGCAGCCCC TCAGAGCGTT

3901 TGCGCTCTGC GTGCAGTGGA TCAGTCCGTG CTGCTGATGA AGCCAGACGC AGAACTGTCT

3961 GCTAGCAGCG TGTATAATCT GCTGCCTGAG AAAGATCTGA CCGGGTTCCC AGGACCTCTG

4021 AACGATCAGG ATGACGAAGA CTGTATTAAT CGCCACAACG TGTATATTAA TGGGATCACA

4081 TACACTCCGG TTTCAAGCAC CAACGAAAAA GATATGTACA GCTTCCTGGA GGACATGGGT

4141 CTGAAAGCGT TTACCAATTC CAAGATCCGG AAACCCCAAG ATGTGCCCAC AGCTCGAGCA

4201 GTATGAAATG CACGGACCTG AGGGTCTGCG TGTGGGCTTT TACGAATCTG ATGTGATGGG

4261 ACGTGGTCAT GCACGTCTGG TTCATGTCGA GGAACCACAC ACCGAAAAGC TTCGTAAATA

4321 CTTCCCTGAG ACCTGGATTT GGGACCTGGT TGTGGTGAAC TCCGCGGGTG TGGCAGAAGT

4381 GGGTGTTACC GTCCCGGATA CTATTACCGA ATGGAAAGCA GGTGCCTTCT GTCTGTCTGA

4441 GGATGCAGGG CTGGGAATCT CCTCTACAGC CTCTCTGCGC GCGTTTCAGC CCTTTTTCGT

4501 CGAACTGACT ATGCCATATA GCGTGATTCG TGGCGAGGCA TTCACTCTGA AAGCTACCGT

4561 GCTGAATTAC CTGCCCAAGT GCATCCGCGT GAGCGTGCAG CTGGAAGCTA GTCCCGCCTT

4621 TCTGGCGGTC CCAGTGGAGA AGGAACAGGC ACCGCACTGC ATTTGTGCTA ACGGCCGGCA

4681 GACTGTTTCC TGGGCCGTCA CCCCCAAATC TCTGGGTAAT GTGAACTTCA CCGTTTCAGC

4741 AGAGGCTCTG GAAAGCCAGG AGCTGTGCGG CACCGAAGTC CCATCCGTGC CTGAGCATGG

4801 TCGCAAAGAT ACAGTCATCA AGCCTCTGCT GGTTGAACCG GAAGGCCTGG AGAAGGAAAC

4861 TACCTTTAAT TCTCTGCTGT GCCCAAGTGG CGGTGAAGTG TCCGAGGAAC TGTCTCTGAA

4921 ACTGCCGCCC AACGTGGTCG AGGAATCTGC CCGTGCGTCA GTTAGCGTCC TGGGGGATAT

4981 TCTGGGAAGT GCCATGCAGA ATACCCAGAA CCTGCTGCAG ATGCCGTATG GCTGTGGCGA

5041 GCAGAATATG GTTCTGTTTG CGCCCAACAT CTATGTCCTG GATTACCTGA ATGAAACACA

5101 GCAGCTGACT CCTGAAATCA AAAGCAAGGC AATCGGGTAT CTGAATACCG GATACCAGCG

5161 GCAGCTGAAC TATAAGCACT ACGACGGCTC CTATTCTACC TTCGGCGAAC GGTACGGTCG

5221 CAATCAGGGG AACACTTGGC TGACCGCCTT TGTGCTGAAA ACCTTTGCCC AGGCTCGCGC

5281 CTATATCTTT ATTGATGAGG CCCATATTAC ACAGGCGCTG ATCTGGCTGT CACAGCGCCA

5341 GAAGGACAAC GGGTGTTTCC GTAGTTCAGG AAGCCTGCTG AACAATGCCA TCAAAGGCGG

5401 CGTCGAGGAT GAAGTGACAC TGAGCGCATA CATTACTATC GCTCTGCTGG AAATCCCTCT

5461 GACAGTGACT CACCCGGTGG TTCGCAATGC TCTGTTTTGC CTGGAAAGTG CATGGAAAAC

5521 AGCTCAGGAA GGCGATCACG GATCACACGT GTATACTAAG GCACTGCTGG CGTACGCATT

5581 CGCTCTGGCC GGCAACCAGG ATAAACGTAA AGAAGTGCTG AAATCACTGA ATGAGGAAGC

5641 AGTTAAAAAG GACAACAGCG TCCACTGGGA ACGGCCGCAG AAACCCAAGG CTCCAGTGGG

5701 TCACTTTTAT GAGCCTCAGG CACCGAGTGC TGAGGTGGAA ATGACCTCAT ATGTTCTGCT

5761 GGCATACCTG ACCGCACAGC CTGCCCCCAC ATCAGAAGAT CTGACAAGCG CCACTAATAT

5821 TGTGAAATGG ATCACCAAGC AGCAGAACGC GCAGGGCGGT TTTAGCTCCA CCCAGGACAC

5881 AGTCGTGGCA CTGCACGCTC TGTCTAAATA TGGGGCAGCT ACCTTCACAC GCACTGGAAA

5941 GGCCGCGCAA GTGACTATTC AGTCTAGTGG CACCTTTTCA AGCAAGTTCC AGGTGGATAA

6001 CAATAACCGT CTGCTGCTGC AGCAGGTGTC CCTGCCCGAA CTGCCAGGCG AGTACTCTAT

6061 GAAAGTCACT GGGGAAGGAT GCGTGTATCT GCAGACCTCC CTGAAATACA ATATTCTGCC

6121 CGAGAAAGAA GAATTTCCAT TCGCACTGGG CGTGCAGACC CTGCCTCAGA CATGCGATGA

6181 ACCGAAGGCT CATACTTCTT TTCAGATCAG TCTGTCAGTG AGCTATACCG GGTCCCGCTC

6241 TGCCAGTAAC ATGGCGATTG TGGATGTGAA AATGGTGAGT GGATTCATCC CTCTGAAACC

6301 GACTGTGAAG ATGCTGGAAC GGAGTAATCA CGTTTCACGC ACCGAGGTCT CCTCTAACCA

6361 TGTGCTGATC TACCTGGATA AAGTGTCCAA TCAGACACTG TCTCTGTTTT TCACTGTGCT

6421 GCAGGATGTC CCCGTGCGTG ACCTGAAACC AGCCATTGTT AAGGTCTATG ATTATTACGA

6481 AACCGACGAG TTCGCGATCG CAGAATACAA CGCGCCGTGC AGCAAAGACC TGGGGAATGC

6541 TGACTACAAG GACGACGACG ACAAGGGGGC AAGCCACCAC CATCACCATC ACTAAGGATC

6601 CAAAATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG

6661 TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA

6721 TTGCATCACA ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC

6781 GATTTCGGCC TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT

6841 CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT

6901 ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA

6961 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA

7021 ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA

7081 CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCTG CCTCTGAGCT ATTCCAGAAG

7141 TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA

7201 TCCATTTTCG GATCTGATCA GCACGTGTTG ACAATTAATC ATCGGCATAG TATATCGGCA

7261 TAGTATAATA CGACAAGGTG AGGAACTAAA CCATGGCCAA GCCTTTGTCT CAAGAAGAAT

7321 CCACCCTCAT TGAAAGAGCA ACGGCTACAA TCAACAGCAT CCCCATCTCT GAAGACTACA

7381 GCGTCGCCAG CGCAGCTCTC TCTAGCGACG GCCGCATCTT CACTGGTGTC AATGTATATC

7441 ATTTTACTGG GGGACCTTGT GCAGAACTCG TGGTGCTGGG CACTGCTGCT GCTGCGGCAG

7501 CTGGCAACCT GACTTGTATC GTCGCGATCG GAAATGAGAA CAGGGGCATC TTGAGCCCCT

7561 GCGGACGGTG CCGACAGGTG CTTCTCGATC TGCATCCTGG GATCAAAGCC ATAGTGAAGG

7621 ACAGTGATGG ACAGCCGACG GCAGTTGGGA TTCGTGAATT GCTGCCCTCT GGTTATGTGT

7681 GGGAGGGCTA ACACGTGCTA CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG

7741 GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC

7801 TGGAGTTCTT CGCCCACCCC AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA

7861 ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT

7921 CCAAACTCAT CAATGTATCT TATCATGTCT GTATACCGTC GACCTCTAGC TAGAGCTTGG

7981 CGTAATCATG GTCATTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA

8041 TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC

8101 TTACCATCTG GCCCCAGCGC TGCGATGATA CCGCGAGAAC CACGCTCACC GGCTCCGGAT

8161 TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA

8221 TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT

8281 AATAGTTTGC GCAACGTTGT TGCCATCGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT

8341 GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG

8401 TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC

8461 GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC

8521 GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG

8581 CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA

8641 ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA

8701 CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT

8761 TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG

8821 GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATATTCT TCCTTTTTCA ATATTATTGA

8881 AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT

8941 AAACAAATAG GGGTCAGTGT TACAACCAAT TAACCAATTC TGAACATTAT CGCG

SEQ ID NO 3: Amino Acid Sequence of Tagged Wild-Type Human A2M

1 MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV

61 SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV

121 MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS

181 FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT

241 ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF

301 YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR

361 QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT

421 VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY

481 ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL

541 LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV

601 DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDDE DCINRHNVYI NGITYTPVSS

661 TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLQQYEMHGP EGLRVGFYES DVMGRGHARL

721 VHVEEPHTET VRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI

781 SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE

841 KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI

901 KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ

961 NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH

1021 YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF

1081 RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH

1141 GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ

1201 APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA

1261 LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG

1321 CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI

1381 VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR

1441 DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNADYKDDD DKGASHHHHHH

SEQ ID NO 4: Amino Acid Sequence of the Acceptor Mutant.

1 MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV

61 SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV

121 MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS

181 FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT

241 ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF

301 YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR

361 QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT

421 VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY

481 ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL

541 LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV

601 DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDDE DCINRHNVYI NGITYTPVSS

661 TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLEQYEMHGP EGLRVGFYES DVMGRGHARL

721 VHVEEPHTEK LRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI

781 SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE

841 KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI

901 KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ

961 NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH

1021 YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF

1081 RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH

1141 GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ

1201 APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA

1261 LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG

1321 CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI

1381 VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR

1441 DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNADYKDDD DKGASHHHHH H

SEQ ID NOs 5-66: Amino Acid Sequences of Variant Bait Regions.

SEQ ID NO 5: LEQYEMHGPE GLRVGKEEEG LGSIPENFFG VSELEGRGSK L

SEQ ID NO 6: LEQYEMHGPE GLRVGIPENF FGVSELEGRG SKEEEGLGSK L

SEQ ID NO 7: LEQYEMHGPE GLRVGSELEG RGSKEEEGLG SIPENFFGVK L

SEQ ID NO 8: LEQYEMHGPE GLRVGKEEEG LGSSELEGRG STAQEAGEGK L

SEQ ID NO 9: LEQYEMHGPE GLRVGIPENF FGVFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 10: LEQYEMHGPE GLRVGKEEEG LGSFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 11: LEQYEMHGPE GLRVGSELEG RGSFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 12: LEQYEMHGPE GLRVGEAIPM SIPFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 13: LEQYEMHGPE GLRVGTAQEA GEGFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 14: LEQYEMHGPE GLRVGVSQEL GQRFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 15: LEQYEMHGPE GLRVGTEGEA RGSFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 16: LEQYEMHGPE GLRVGTSEDL VVQFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 17: LEQYEMHGPE GLRVGEGEGE GEGFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 18: LEQYEMHGPE GLRVGGEEGV EEGFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 19: LEQYEMHGPE GLRVGGARGL EGFYESDVMG RGHARLVHVE EPHTKL

SEQ ID NO 20: LEQYEMHGPE GLRVGGPPGL APGFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 21: LEQYEMHGPE GLRVGGEPEG AKGFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 22: LEQYEMHGPE GLRVGEEEGG GFYESDVMGR GHARLVHVEE PHTKL

SEQ ID NO 23: LEQYEMHGPE GLRVGGYPGS SRGFYESDVM GRGHARLVHV EEPHTKLL

SEQ ID NO 24: LEQYEMHGPE GLRVGGARGL EGGFAGLPNG GEEGVEEGKL

SEQ ID NO 25: LEQYEMHGPE GLRVGESESE GGGGGSLLGE FEVEGGFAGL PNGKL

SEQ ID NO 26: LEQYEMHGPE GLRVGGFKEG VEGEIEEGGG FKEGVEGKL

SEQ ID NO 27: LEQYEMHGPE GLRVGESESE GGFAGLPNGK EEEGLGSIPE NFFGVKL

SEQ ID NO 28: LEQYEMHGPE GLRVGIPENF FGVTSEDLVV QEAIPMSIPK L

SEQ ID NO 29: LEQYEMHGPE GLRVGEAIPM SIPTSEDLVV QIPENFFGVK L

SEQ ID NO 30: LEPAGAARGE SESEGGFFGF PIGERESTGG DRGLPIGENE AGGKL

SEQ ID NO 31: LETEGRGERE AQGEFPEVEG EEEGGGPEKE TGGEREAQGK L

SEQ ID NO 32: LEARGLEGGG GGSLLGGYPG SSRGGFKEGV EGG PAG A ARC KL

SEQ ID NO 33: LEPGLAPGGE EGVEEGGPEE GVEEGGFKEG VEGEPESSGK L

SEQ ID NO 34: LEEGEARGST AQEAGEGPKE QELGQRSELE GRGSPVSQEL GQRKL

SEQ ID NO 35: LEAQEAGEGK EEEGLGSPVS QELGQRSELE GRGSPTEGEA RGSKL

SEQ ID NO 36: LEEEEGLGSK EEEGLGSPKE EEGLGSKEEE GLGSPKEEEG LGSKL

SEQ ID NO 37: LEELEGRGSK EEEGLGSIPE NFFGVFYESD VMGRGHARLV HVEEPHTKL

SEQ ID NO 38: LEENFFGVTE GEARGSPTSE DLVVQKEEEG LGSEAIPMSI PKL

SEQ ID NO 39: LEIPMSIPKE EEGLGSIPEN FFGVTEGEAR GSPTSEDLVV QKL

SEQ ID NO 40: LELQQYEMHG PEGLRVGEAI PMSIPIPENF FGVKEEEGLG SKL

SEQ ID NO 41: LEEEGVEEGK EEEGLGSGPA GAARGSELEG RGSPTEGEAR GSKL

SEQ ID NO 42: LEPESSGEAI PMSIPTSEDL VVQIPENFFG VEAEGTGGER GVLGKL

SEQ ID NO 43: LEGGGSLLGE PEPEGEREAQ GGVEGVELGG FKEGVEGEQE GRGKL

SEQ ID NO 44: LESQELGQRE SESEGSELEG RGSGFKEGVE GKEEEGLGSG FFGFPIGKL

SEQ ID NO 45: LEQYEMHGPK EEEGLGSSEL EGRGSEAIPM SIPTIPENFF GVVEEPHTKL

SEQ ID NO 46: LEQYEMHGPS ELEGRGSIPE NFFGVEAIPM SIPTSEDLVV QIVEEPHTKL

SEQ ID NO 47: LEQYEMHGPE GEGEGEGIPE NFFGVSEDLV VQISELEGRG SVEEPHTKL

SEQ ID NO 50: LEQYEMHGPI PENFFGVSEL EGRGSEAIPM SIPTEGEGEG EGVEEPHTKL

SEQ ID NO 51: LEQYEMHGPS ELEGRGSEAI PMSIPTKEEE GLGSIPENFF GVVEEPHTKL

SEQ ID NO 52: LEQYEMHGPE AIPMSIPTEG EGEGEGIPEN FFGVSEDLVV QIVEEPHTKL

SEQ ID NO 53: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVEAIPMSI PTVEEPHTKL

SEQ ID NO 54: LEQYEMHGPE GEGEGEGISE DLVVQIPENF FGVKEEEGLG SVEEPHTKL

SEQ ID NO 55: LEQYEMHGPE GEGEGEGIPE NFFGVSELEG RGSSEDLVVQ IVEEPHTKL

SEQ ID NO 56: LEQYEMHGPI PENFFGVEGE GEGESELEGR GSSEDLVVQI VEEPHTKL

SEQ ID NO 57: LEQYEMHGPS ELEGRGSIPE NFFGVKEEEG LGSSEDLVVQ IVEEPHTKL

SEQ ID NO 58: LEQYEMHGPI PENFFGVSEL EGRGSSEDLV VQIKEEEGLG SVEEPHTKL

SEQ ID NO 59: LEQYEMHGPK EEEGLGSIPE NFFGVSELEG RGSEGEGEGE GVEEPHTKL

SEQ ID NO 60: LEQYEMHGPS EDLVVQIKEE EGLGSIPENF FGVSELEGRG SVEEPHTKL

SEQ ID NO 61: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVKEEEGLG SVEEPHTKL

SEQ ID NO 62: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVEAIPMSI PTEPHTKL

SEQ ID NO 63: LEQYEMHGPE GEGEGEGIPE NFFGVEAIPM SIPTSELEGR GSEPHTKL

SEQ ID NO 64: LEQYEMHGPE AIPMSIPTSE LEGRGSIPEN FFGVEGEGEG EGEPHTKL

SEQ ID NO 65: LEQYEMHGPS ELEGRGSIPE NFFGVEGEGE GEGKEEEGLG SVEEPHTKL

SEQ ID NO 66: LEQYEMHGPI PENFFGVSED LVVQIEGEGE GEGEAIPMSI PTEPHTKL

What is claimed is:
 1. A method for the treatment or prophylaxis of achronic wound in a mammalian subject, comprising topically applying tothe chronic wound an effective amount of a composition comprising arecombinant alpha-2-macroglobulin polypeptide (A2M) comprising anon-natural bait region that replaces a wild-type A2M bait region. 2.The method of claim 1, wherein activity of a protease of the mammaliansubject is reduced by at least 10% at a site of topical application. 3.The method of claim 1, wherein activity of a protease of the mammaliansubject is reduced at a site of topical application within an hour ofthe topically applying.
 4. The method of claim 1, whereinproinflammatory activity of the mammalian subject is reduced by at least10% at a site of topical application.
 5. The method of claim 1, whereinthe mammalian subject is a human.
 6. The method of claim 1, wherein thechronic wound results from a cosmetic procedure.
 7. The method of claim1, wherein the composition is on a wound dressing.
 8. The method ofclaim 1, wherein severity of the chronic wound is reduced, size of thechronic wound is reduced, infection of the chronic wound is reduced,bleeding of the chronic wound is reduced, or healing rate of the chronicwound is increased in the mammalian subject.
 9. The method of claim 1,wherein the chronic wound is a sore or an ulcer.
 10. The method of claim9, wherein the chronic wound is an ulcer selected from the groupconsisting of a venous ulcer, a diabetic pressure ulcer, a stasis ulcer,a venous stasis ulcer, a diabetic foot ulcer, an arterial insufficiencyulcers, a burn ulcer, a traumatic ulcer, and combinations thereof. 11.The method of claim 9, wherein the chronic wound is a pressure sore. 12.The method of claim 1, wherein the composition is a liquid.
 13. Themethod of claim 1, wherein the A2M is a recombinant A2M characterized byat least a 10% enhanced inhibition of a protease compared an inhibitionof the protease by an equivalent amount of a wild-type A2M, wherein thenon-natural bait region comprises a recognition site for a protease thatis not present in a wild-type A2M.
 14. The method of claim 13, whereinthe non-natural bait region comprises a plurality of recognition sitesfor one or more proteases.
 15. The method of claim 13, wherein therecombinant A2M is characterized by at least a 10% enhanced inhibitionof two or more different proteases compared to an inhibition of the twoor more different proteases by an equivalent amount of a wild-type A2M.16. The method of claim 13, wherein the protease is selected from thegroup consisting of a serine protease, a threonine protease, a cysteineprotease, an aspartate protease, a glutamic acid protease, ametalloprotease, and combinations thereof.
 17. The method of claim 13,wherein the protease is selected from the group consisting of matrixmetalloprotease 1 (MMP1), MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11,MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21,MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28; A Disintegrin andMetalloproteinase with Thrombospondin Motifs protease 1 (ADAMTS1),ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9,ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17,ADAMTS18, ADAMTS19, ADAMTS20, chymotrypsin, trypsin, elastase, acompliment factor; a clotting factor, thrombin, plasmin, subtilisin,neprilysin, thermolysin, Pregnancy-associated plasma protein A; Bonemorphogenetic protein 1, lysostaphin, insulin degrading enzyme,ZMPSTE24, acetylcholinesterase, and combinations thereof.
 18. The methodof claim 1, wherein the composition does not elicit an immune responseby the mammalian subject when topically applied to the chronic wound.19. A wound dressing comprising an effective amount of a composition forthe treatment or prophylaxis of a chronic wound in a mammalian subject,wherein the composition comprises a recombinant alpha-2-macroglobulinpolypeptide (A2M) comprising a non-natural bait region that replaces awild-type A2M bait region.